DNA encoding a plant deoxyhypusine synthase, a plant eukaryotic initiation factor 5A, transgenic plants and a method for controlling senescence programmed and cell death in plants

ABSTRACT

Regulation of expression of programmed cell death, including senescence, in plants is achieved by integration of a gene or gene fragment encoding senescence-induced deoxyhypusine synthase, senescence-induced elF-5A or both into the plant genome in antisense orientation. Plant genes encoding senescence-induced deoxyhypusine synthase and senescence-induced elF-5A are identified and the nucleotide sequences of each, alone and in combination are used to modify senescence in transgenic plants.

[0001] This application is a continuation-in-part application of Ser.No. 09/348,675, filed Jul. 6, 1999.

FIELD OF THE INVENTION

[0002] The present invention relates to polynucleotides which encodeplant polypeptides that exhibit senescence-induced expression. Theinvention also relates to transgenic plants containing thepolynucleotides in antisense orientation and methods for controllingprogrammed cell death, including senescence, in plants. Moreparticularly, the present invention relates to a senescence inducedplant deoxyhypusine synthase gene and a senescence-induced elF-5A genewhose expressions are induced by the onset of programmed cell death,including senescence, and the use of the deoxyhypusine synthase gene andelF-5A gene, alone or in combination, to control programmed cell deathand senescence in plants.

DESCRIPTION OF THE PRIOR ART

[0003] Senescence is the terminal phase of biological development in thelife of a plant. It presages death and occurs at various levels ofbiological organization including the whole plant, organs, flowers andfruit, tissues and individual cells.

[0004] The onset of senescence can be induced by different factors bothinternal and external. Senescence is a complex, highly regulateddevelopmental stage in the life of a plant or plant tissue, such asfruit, flowers and leaves. Senescence results in the coordinatedbreakdown of cell membranes and macromolecules and the subsequentmobilization of metabolites to other parts of the plant.

[0005] In addition to the programmed senescence which takes place duringnormal plant development, death of cells and tissues and ensuingremobilization of metabolites occurs as a coordinated response toexternal, environmental factors. External factors that induce prematureinitiation of senescence, which is also referred to as necrosis orapoptosis, include environmental stresses such as temperature, drought,poor light or nutrient supply, as well as pathogen attack. Plant tissuesexposed to environmental stress also produce ethylene, commonly known asstress ethylene (Buchanan-Wollaston, V., 1997, J. Exp. Botany,48:181-199; Wright, M., 1974, Plant, 120:63-69). Ethylene is known tocause senescence in some plants.

[0006] Senescence is not a passive process, but, rather, is an activelyregulated process that involves coordinated expression of specificgenes. During senescence, the levels of total RNA decrease and theexpression of many genes is switched off (Bate et al., 1991, J. Exper.Botany, 42, 801-11; Hensel et al., 1993, The Plant Cell, 5, 553-64).However, there is increasing evidence that the senescence processdepends on de novo transcription of nuclear genes. For example,senescence is blocked by inhibitors of mRNA and protein synthesis andenucleation. Molecular studies using mRNA from senescing leaves andgreen leaves for in vitro translation experiments show a changed patternof leaf protein products in senescing leaves (Thomas et al, 1992, J.Plant Physiol., 139, 403-12). With the use of differential screening andsubtractive hybridization techniques, many cDNA clones representingsenescence-induced genes have been identified from a range of differentplants, including both monocots and dicots, such as Arabidopsis, maize,cucumber, asparagus, tomato, rice and potato. Identification of genesthat are expressed specifically during senescence is hard evidence ofthe requirement for de novo transcription for senescence to proceed.

[0007] The events that take place during senescence appear to be highlycoordinated to allow maximum use of the cellular components beforenecrosis and death occur. Complex interactions involving the perceptionof specific signals and the induction of cascades of gene expressionmust occur to regulate this process. Expression of genes encodingsenescence related proteins is probably regulated via common activatorproteins that are, in turn, activated directly or indirectly by hormonalsignals. Little is known about the mechanisms involved in the initialsignaling or subsequent co-ordination of the process.

[0008] Coordinated gene expression requires factors involved intranscription and translation, including initiation factors. Translationinitiation factor genes have been isolated and characterized in avariety of organisms, including plants. Eukaryotic translationinitiation factor 5A (elF-5A) is an essential protein factorapproximately 17 KDa in size, which is involved in the initiation ofeukaryotic cellular protein synthesis. It is characterized by thepresence of hypusine [N-(4-amino-2-hydroxybutyl) lysine], a uniquemodified amino acid, known to be present only in elF-5A. Hypusine isformed post-translationally via the transfer and hydroxylation of thebutylamino group from the polyamine, spermidine, to the side chain aminogroup of a specific lysine residue in elF-5A. Activation of elF-5Ainvolves transfer of the butylamine residue of spermidine to the lysineof elF-5A, forming hypusine and activating elF-5A. In eukaryotes,deoxyhypusine synthase (DHS) mediates the post-translational synthesisof hypusine in elF-5A. A corresponding DHS gene has not been identifiedin plants, however, it is known that plant elF-5A contains hypusine. Thehypusine modification has been shown to be essential for elF-5A activityin vitro using a methionyl-puromycin assay.

[0009] Hypusine is uniquely present in elF-5A and is found in alleukaryotes, some archaebacteria (which appear to be related toeukaryota), but not in eubacteria. Moreover, the amino acid sequence ofelF-5A is highly conserved, especially in the region surrounding thehypusine residue, suggesting that elF-5A and its activating protein,deoxyhypusine synthase, execute fundamentally important steps ineukaryotic cell physiology (Joe et al., JBC, 270:22386-22392, 1995).elF-5A has been cloned from human, alfalfa, slime mold, Neurosporacrassa, tobacco and yeast. It was originally identified as a generaltranslation initiation factor based on its isolation from ribosomes ofrabbit reticulocyte lysates and its in vitro activity in stimulatingmethionine-puromycin synthesis. However, more recent data indicate thatelF-5A is not a translation initiation factor for global proteinsynthesis, but rather serves to facilitate the translation of specificsubsets of mRNA populations. For example, there is strong evidence fromexperiments with animal cells and yeast that one or more isoforms ofelF-5A play an essential role in mediating the translation of a subsetof mRNAs involved in cell proliferation. There are two isoforms inyeast, and if both genes are silenced the cells are unable to divide(Park et al., Biol. Signals, 6:115-123, 1997). Similarly, silencing theexpression of yeast deoxyhypusine synthase, which activates elF-5A,blocks cell division. Indeed, inhibitors of deoxyhypusine synthase havebeen developed that are likely to have importance in the therapy ofhyperproliferative conditions (Wolff, et al., JBC, 272:15865-15871,1997). Other studies have indicated that another isoform of elF-5A isessential for Rev function in HIV-1 replication or Rex function in HTLVV replication (Park, et al., Biol. Signals, 6:115-123, 1997). There arealso at least two expressed elF-5A genes in tobacco. Gene-specificprobes indicate that although they are both expressed in all tissuesexamined, each gene has a distinctive expression pattern, presumablyregulating the translation of specific transcripts (Chamot, et al., Nuc.Acids Res., 20:625-669, 1992).

[0010] Deoxyhypusine synthase has been purified from rat testis, HeLacells, Neurospora crassa and yeast. The amino acid sequence ofdeoxyhypusine synthase is highly conserved, and the enzymes fromdifferent species share similar physical and catalytic properties anddisplay cross-species reactivities with heterologous elF-5A precursors(Park, et al., 6 Biol. Signals, 6:115-123, 1997).

[0011] Plant polyamines have been implicated in a wide variety ofphysiological effects including floral induction, embryogenesis,pathogen resistance, cell growth, differentiation and division (Evans etal., 1989, Annu. Rev. Plant Physiol. Plant Mol. Biol., 40, 235-269; andGalston, et al., 1990, Plant Physiol., 94, 406-10). It has beensuggested that elF-5A is the intermediary through which polyamines exerttheir effects (Chamot et al., 1992, Nuc. Acids Res., 20(4), 665-69).

[0012] Two genes encoding isoforms of elF-5A from Nicotiana have beenidentified (NelF-5A1 and NelF-5A2) (Chamot et al., 1992, Nuc. AcidsRes., 20(4), 665-69). The genes were shown to be very similar. However,they display differential patterns of expression. One gene appears to beconstitutively expressed at the mRNA level, while the expression patternof the other correlates with the presence or absence of photosyntheticactivity. Based on gene structure and genomic Southern mapping it hasbeen suggested that there is a multigene family of NelF-5A genes intobacco. It is likely that there is an elF-5A isoform that regulatestranslation of a subset of senescence/necrosis specific mRNAtranscripts.

[0013] Presently, there is no widely applicable method for controllingthe onset of programmed cell death (including senescence) caused byeither internal or external, e.g., environmental stress, factors. It is,therefore, of interest to develop senescence modulating technologiesthat are applicable to all types of plants and that are effective at theearliest stages in the cascade of events leading to senescence.

SUMMARY OF THE INVENTION

[0014] This invention is based on the discovery and cloning of a fulllength cDNA clone encoding a tomato senescence-induced deoxyhypusinesynthase (DHS), as well as full length senescence-induced DHS cDNAclones from Arabidopsis leaf and carnation petal. The nucleotidesequences and corresponding amino acid sequences are disclosed herein.

[0015] The invention is also based, in part, on the discovery andcloning of full length cDNA clones encoding a senescence-induced elF-5Agene from tomato, Arabidopsis and carnation. The nucleotide sequence andcorresponding amino acid sequence of each of the elF-5A cDNA clones aredisclosed herein.

[0016] The present invention provides a method for genetic modificationof plants to control the onset of senescence, either age-relatedsenescence or environmental stress-induced senescence. Thesenescence-induced DHS nucleotide sequences of the invention, fragmentsthereof, or combinations of such fragments, are introduced into a plantcell in reverse orientation to inhibit expression of the endogenoussenescence-induced DHS gene, thereby reducing the level of endogenoussenescence-induced

[0017] DHS protein, and reducing and/or preventing activation of elF-5Aand ensuing expression of the genes that mediate senescence.

[0018] In another aspect of the invention, the senescence-induced elF-5Anucleotide sequences of the invention, fragments thereof, orcombinations of such fragments, are introduced into a plant cell inreverse orientation to inhibit expression of the endogenoussenescence-induced elF-5A gene, and thereby reduce the level ofendogenous senescence-induced elF-5A protein, and reduce and/or preventensuing expression of the genes that mediate senescence. Alternatively,both DHS sequences and elF-5A sequences can be used together to reducethe levels of endogenous DHS and elF-5A proteins

[0019] In yet another aspect, the present invention is directed to amethod for genetic modification of plants to control the onset ofsenescence, either age-related senescence or environmentalstress-induced senescence via the introduction into a plant cell of acombination of senescence-induced elF-5A nucleotide sequences of theinvention and senescence-induced DHS nucleotide sequences of theinvention in reverse orientation to inhibit expression of the endogenoussenescence-induced elF-5A gene and senescence-induced DHS gene, therebyreducing the level of endogenous senescence-induced DHS protein, andreducing and/or preventing activation of elF-5A and ensuing expressionof the genes that mediate senescence.

[0020] Using the methods of the invention, transgenic plants aregenerated and monitored for growth, development and either natural orprematurely-induced senescence. Plants or detached parts of plants(e.g., cuttings, flowers, vegetables, fruits, seeds or leaves)exhibiting prolonged life or shelf life, (e.g., extended life offlowers, reduced fruit or vegetable spoilage, enhanced biomass,increased seed yield, reduced seed aging and/or reduced yellowing ofleaves) due to reduction in the level of senescence-induced DHS,senescence-induced elF-5A or both are selected as desired productshaving improved properties including reduced leaf yellowing, reducedpetal abscission, reduced fruit and vegetable spoilage during shippingand storage. These superior plants are propagated. Similarly, plantsexhibiting increased resistance to environmental stress, e.g., decreasedsusceptibility to low temperature (chilling), drought, infection, etc.,and/or increased resistance to pathogens, are selected as superiorproducts.

[0021] In one aspect, the present invention is directed to an isolatedDNA molecule encoding senescence-induced DHS, wherein the DNA moleculehybridizes with SEQ ID NO:1, or a functional derivative of the isolatedDNA molecule which hybridizes with SEQ ID NO:1. In one embodiment ofthis aspect of the invention, the isolated DNA molecule has thenucleotide sequence of SEQ ID NO:1, i.e., 100% complementarity (sequenceidentity) to SEQ ID NO:1.

[0022] The present invention also is directed to an isolated DNAmolecule encoding senescence-induced DHS, wherein the DNA moleculehybridizes with SEQ ID NO:9, or a functional derivative of the isolatedDNA molecule which hybridizes with SEQ ID NO:9. In one embodiment ofthis aspect of the invention, the isolated DNA molecule has thenucleotide sequence of SEQ ID NO:9, i.e., 100% complementarity (sequenceidentity) to SEQ ID NO:9.

[0023] The present invention also is directed to an isolated DNAmolecule encoding senescence-induced elF-5A, wherein the DNA moleculehybridizes with SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15 or a functionalderivative of the isolated DNA molecule which hybridizes with SEQ IDNO:11, SEQ ID NO:13, SEQ ID NO:15. In one embodiment of this aspect ofthe invention, the isolated DNA molecule has the nucleotide sequence ofSEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:15, i.e., 100% complementarity(sequence identity) to SEQ ID NO:11, SEQ ID NO:13 or SEQ ID NO:15.

[0024] In another embodiment of the invention, there is provided anisolated protein encoded by a DNA molecule as described herein above, ora functional derivative thereof. A preferred protein has the amino acidsequence of SEQ ID NO:2, or is a functional derivative thereof. Anotherpreferred protein has the amino acid sequence of SEQ ID NO:10, or is afunctional derivative thereof. Other preferred proteins of the inventionhave the amino acid sequence of SEQ ID NO:12, SEQ ID NO:14 or SEQ ID NO:16.

[0025] Also provided herein is an antisense oligonucleotide orpolynucleotide encoding an RNA molecule which is complementary to acorresponding portion of an RNA transcript of a DNA molecule describedherein above, wherein the oligonucleotide or polynucleotide hybridizeswith the RNA transcript such that expression of endogenoussenescence-induced DHS is altered. In another embodiment of this aspectof the invention, the antisense oligonucleotide or polynucleotide is anRNA molecule that hybridizes to a corresponding portion of an RNAtranscript of a DNA molecule described herein above, such thatexpression of endogenous senescence-induced elF-5A is altered. Theantisense oligonucleotide or polynucleotide can be full length orpreferably has about six to about 100 nucleotides.

[0026] The antisense oligonucleotide or polynucleotide may besubstantially complementary to a corresponding portion of one strand ofa DNA molecule encoding senescence-induced DHS, wherein the DNA moleculeencoding senescence-induced DHS hybridizes with SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO: 9, or with a combination thereof, or is substantiallycomplementary to at least a corresponding portion of an RNA sequenceencoded by the DNA molecule encoding senescence-induced DHS. In oneembodiment of the invention, the antisense oligonucleotide orpolynucleotide is substantially complementary to a corresponding portionof one strand of the nucleotide sequence SEQ ID NO:1, SEQ ID NO:5, SEQID NO:9 or with a combination thereof, or the RNA transcript transcribedfrom SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:9 or with a combinationthereof. In another embodiment, the antisense oligonucleotide issubstantially complementary to a corresponding portion of the 5′non-coding portion or 3′ portion of one strand of a DNA moleculeencoding senescence-induced DHS, wherein the DNA molecule hybridizeswith SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:9 or with a combinationthereof.

[0027] Alternatively, the antisense oligonucleotide or polynucleotidemay be substantially complementary to a corresponding portion of onestrand of a DNA molecule encoding senescence-induced elF-5A, wherein theDNA molecule encoding senescence-induced elF-5A hybridizes with SEQ IDNO:11, SEQ ID NO:13, SEQ ID NO:15, or any combination thereof, or issubstantially complementary to at least a corresponding portion of anRNA sequence transcribed from SEQ ID NO:11, SEQ ID NO:13 or SEQ IDNO:15. In one embodiment of the invention, the antisense oligonucleotideor polynucleotide is substantially complementary to a correspondingportion of one strand of the nucleotide sequence SEQ ID NO:11, SEQ IDNO:13, SEQ ID NO:15 or a combination thereof, or the RNA transcriptencoded is substantially complementary to a corresponding portion of anRNA sequence encoded by a DNA molecule encoding senescence-inducedelF-5A. In another embodiment, the antisense oligonucleotide issubstantially complementary to a corresponding portion of the 5′non-coding region or 3′ region of one strand of a DNA molecule encodingsenescence-induced elF-5A, wherein the DNA molecule hybridizes with SEQID NO:11, SEQ ID NO:13, SEQ ID NO:15 or a combination thereof.

[0028] The invention is further directed to a vector for transformationof plant cells, comprising

[0029] (a) an antisense oligo- or polynucleotide substantiallycomplementary to (1) a corresponding portion of one strand of a DNAmolecule encoding senescence-induced DHS, wherein the DNA moleculeencoding senescence-induced DHS hybridizes with SEQ ID NO:1, SEQ ID NO:5or SEQ ID NO:9, or (2) a corresponding portion of an RNA sequenceencoded by the DNA molecule encoding senescence-induced DHS; and

[0030] (b) regulatory sequences operatively linked to the antisenseoligo- or polynucleotide such that the antisense oligo- orpolynucleotide is expressed in a plant cell into which it istransformed.

[0031] The invention is further directed to a vector for transformationof plant cells, comprising

[0032] (a) an antisense oligo- or polynucleotide substantiallycomplementary to (1) a corresponding portion of one strand of a DNAmolecule encoding senescence-induced elF-5A, wherein the DNA moleculeencoding senescence-induced elF-5A hybridizes with SEQ ID NO:11, SEQ IDNO:13, SEQ ID NO:15 or (2) a corresponding portion of an RNA sequenceencoded by the DNA molecule encoding senescence-induced elF-5A; and

[0033] (b) regulatory sequences operatively linked to the antisenseoligo- or polynucleotide such that the antisense oligo- orpolynucleotide is expressed in a plant cell into which it istransformed.

[0034] The regulatory sequences include a promoter functional in thetransformed plant cell, which promoter may be inducible or constitutive.Optionally, the regulatory sequences include a polyadenylation signal.

[0035] The invention also provides a plant cell transformed with avector or combination of vectors as described above, a plantlet ormature plant generated from such a cell, or a plant part of such aplantlet or plant.

[0036] The present invention is further directed to a method ofproducing a plant having a reduced level of senescence-induced DHS,senescence-induced elF-5A or both compared to an unmodified plant,comprising:

[0037] (1) transforming a plant with a vector or combination of vectorsas described above;

[0038] (2) allowing the plant to grow to at least a plantlet stage;

[0039] (3) assaying the transformed plant or plantlet for alteredsenescence-induced DHS activity and/or elF-5A activity and/or alteredsenescence and/or altered environmental stress-induced senescence and/orpathogen-induced senescence and/or ethylene-induced senescence; and

[0040] (4) selecting and growing a plant having alteredsenescence-induced DHS activity and/or reduced elF-5A and/or alteredsenescence and/or altered environmental stress-induced senescence and/oraltered pathogen-induced senescence and/or ethylene-induced senescencecompared to a non-transformed plant.

[0041] Plants produced as above, or progeny, hybrids, clones or plantparts preferably exhibit reduced senescence-induced DHS expression,reduced senescence-induced elF-5A activity, or both and delayedsenescence and/or delayed stress-induced senescence and/orpathogen-induced senescence and/or ethylene-induced senescence.

[0042] This invention is further directed to a method of inhibitingexpression of endogenous senescence-induced DHS in a plant cell, saidmethod comprising:

[0043] (1) integrating into the genome of a plant a vector comprising

[0044] (A) an antisense oligo- or polynucleotide complementary to (I) atleast a portion of one strand of a DNA molecule encoding endogenoussenescence-induced DHS, wherein the DNA molecule encoding the endogenoussenescence-induced DHS hybridizes with SEQ ID NO:1, SEQ ID NO:5 and/orSEQ ID NO.9, or (ii) at least a portion of an RNA sequence encoded bythe endogenous senescence-induced DHS gene; and

[0045] (B) regulatory sequences operatively linked to the antisenseoligo- or polynucleotide such that the antisense oligo- orpolynucleotide is expressed; and

[0046] (2) growing said plant, whereby said antisense oligo- orpolynucleotide is transcribed and the transcript binds to saidendogenous RNA whereby expression of said senescence-induced DHS gene isinhibited.

[0047] This invention is further directed to a method of inhibitingexpression of endogenous senescence-induced elF-5A in a plant cell, saidmethod comprising:

[0048] (1) integrating into the genome of a plant a vector comprising

[0049] (A) an antisense oligo- or polynucleotide complementary to (I) acorresponding portion of one strand of a DNA molecule encodingendogenous senescence-induced elF-5A, wherein the DNA molecule encodingthe endogenous senescence-induced elF-5A hybridizes with SEQ ID NO:11,SEQ ID NO:15, SEQ ID NO:17 or a combination thereof, or (ii) at least aportion of an RNA sequence encoded by the endogenous senescence-inducedelF-5A gene; and

[0050] (B) regulatory sequences operatively linked to the antisenseoligo- or polynucleotide such that the antisense oligo- orpolynucleotide is expressed; and

[0051] (2) growing said plant, whereby said antisense oligo- orpolynucleotide is transcribed and the transcript binds to saidendogenous RNA whereby expression of said senescence-induced elF-5A geneis inhibited.

BRIEF DESCRIPTION OF THE DRAWINGS

[0052]FIG. 1 depicts the nucleotide sequence of the senescence-inducedtomato leaf DHS cDNA sequence (SEQ ID NO:1) and the derived amino acidsequence (SEQ ID NO. 2) obtained from a tomato leaf cDNA library.

[0053]FIG. 2A depicts the nucleotide sequence of an Arabidopsis DHS geneobtained by aligning the tomato DHS sequence with unidentified genomicsequences in the Arabidopsis gene bank(http://genome-www.stanford.edu/Arabidopsis/) (SEQ ID NO:5). The gapsbetween amino acid sequences are predicted introns. FIG. 2B depicts thederived Arabidopsis DHS amino acid sequence (SEQ ID NO:6). FIG. 2Cdepicts the nucleotide sequence of a 600 base pair senescence-inducedArabidopsis DHS cDNA obtained by PCR. FIG. 2D depicts the derived aminoacid sequence of the senescence-induced Arabidopsis DHS cDNA fragment.

[0054]FIG. 3 is an alignment of the derived full length tomato leafsenescence-induced DHS amino acid sequence (SEQ ID NO. 2) and thederived full length Arabidopsis senescence-induced DHS amino acidsequence with sequences of DHS proteins of human, yeast, fungi, andArchaeobacteria. Identical amino acids among three or four of thesequences are boxed.

[0055]FIG. 4 is a restriction map of the tomato DHS cDNA.

[0056]FIG. 5 is a Southern blot of genomic DNA isolated from tomatoleaves and probed with ³²P-dCTP-labeled full length tomatosenescence-induced DHS cDNA.

[0057]FIG. 6 is a Northern blot of RNA isolated from tomato flowers atdifferent stages of development. FIG. 6A is the ethidium bromide stainedgel of total RNA. Each lane contains 10 μg RNA. FIG. 6B is anautoradiograph of the Northern blot probed with ³²P-dCTP-labeled fulllength tomato senescence-induced DHS cDNA.

[0058]FIG. 7 is a Northern blot of RNA isolated from tomato fruit atvarious stages of ripening that was probed with ³²P-dCTP-labelled fulllength tomato senescence-induced DHS cDNA. Each lane contains 10 μg RNA.

[0059]FIG. 8 is a Northern blot of RNA isolated from tomato leaves thathad been drought-stressed by treatment with 2 M sorbitol for six hours.Each lane contains 10 μg RNA. The blot was probed with ³²P-dCTP-labelledfull length tomato senescence-induced DHS cDNA.

[0060]FIG. 9 is a Northern blot of RNA isolated from tomato leaves thathad been exposed to chilling temperature. FIG. 9A is the ethidiumbromide stained gel of total RNA. Each lane contained 10 μg RNA. FIG. 9Bis an autoradiograph of the Northern blot probed with ³²P-dCTP-labelledfull length tomato senescence-induced DHS cDNA. FIG. 9C showscorresponding leakage data measured as conductivity of leaf diffusates.

[0061]FIG. 10 is the carnation DHS full-length (1384 base pairs) cDNAclone nucleotide sequence (SEQ ID NO: 9), not including the PolyA tailand 5′ end non-coding region. The derived amino acid sequence is shownbelow the nucleotide sequence (373 amino acids). (SEQ ID NO:10)

[0062]FIG. 11 is a Northern blot of total RNA from senescing Arabidopsisleaves probed with ³²P-dCTP-labelled full-length Arabidopsissenescence-induced DHS cDNA. The autoradiograph is at the top, theethidium stained gel below.

[0063]FIG. 12 is a Northern blot of total RNA isolated from petals ofcarnation flowers at various stages. The blot was probed with³²P-dCTP-labelled full-length carnation senescence-induced DHS cDNA. Theautoradiograph is at the top, the ethidium stained gel below.

[0064]FIG. 13 is the nucleotide (top) (SEQ ID NO:11) and derived aminoacid (bottom) (SEQ ID NO:12) sequence of the tomato fruitsenescence-induced elF-5A gene.

[0065]FIG. 14 is the nucleotide (top) (SEQ ID NO:13) and derived aminoacid (bottom) (SEQ ID NO:14) sequence of the carnationsenescence-induced elF-5A gene.

[0066]FIG. 15 is the nucleotide (top) (SEQ ID NO:15) and derived aminoacid (bottom) (SEQ ID NO:16) sequence of the Arabidopsissenescence-induced elF-5A gene.

[0067]FIG. 16 is a Northern blot of total RNA isolated from leaves ofArabidopsis plants at various developmental stages. The blot was probedwith ³²P-dCTP-labelled full-length Arabidopsis senescence-induced DHScDNA and full-length senescence-induced elF-5A. The autoradiograph is atthe top, the ethidium stained gel below.

[0068]FIG. 17 is a Northern blot of total RNA isolated from tomato fruitat breaker (BK), red-firm (RF) and red-soft (RS) stages of development.The blot was probed with ³²P-dCTP-labelled full-lengthsenescence-induced DHS cDNA and full-length senescence-induced elF-5A.DHS and elF-5A are up-regulated in parallel in red-soft fruit coincidentwith fruit ripening. The autoradiograph is at the top, the ethidiumstained gel below.

[0069]FIG. 18 is a Northern blot of total RNA isolated from leaves oftomato that were treated with sorbitol to induce drought stress. C iscontrol; S is sorbitol treated. The blot was probed with³²P-dCTP-labelled full-length senescence-induced DHS cDNA andfull-length senescence-induced elF-5A. Both elF-5A and DHS areup-regulated in response to drought stress. The autoradiograph is at thetop, the ethidium stained gel below.

[0070]FIG. 19 is a Northern blot of total RNA isolated from flower budsand open senescing flowers of tomato plants. The blot was probed with³²P-dCTP-labelled full-length senescence-induced DHS cDNA andfull-length senescence-induced elF-5A. Both elF-5A and DHS areup-regulated in open/senescing flowers. The autoradiograph is at thetop, the ethidium stained gel below.

[0071]FIG. 20 is a Northern blot of total RNA isolated fromchill-injured tomato leaves. The blot was probed with ³²P-dCTP-labelledfull-length senescence-induced DHS cDNA and full-lengthsenescence-induced elF-5A. Both elF-5A and DHS are up-regulated with thedevelopment of chilling injury during rewarming The autoradiograph is atthe top, the ethidium stained gel below.

[0072]FIG. 21 is a photograph of 3.1 week old Arabidopsis wild-type(left) and transgenic plants expressing the 3′-end of the senescence DHSgene (sequence shown in FIG. 36) in antisense orientation showingincreased leaf size in the transgenic plants.

[0073]FIG. 22 is a photograph of 4.6 week old Arabidopsis wild-type(left) and transgenic plants expressing the 3′-end of the senescence DHSgene (sequence shown in FIG. 36) in antisense orientation showingincreased leaf size in the transgenic plants.

[0074]FIG. 23 is a photograph of 5.6 week old Arabidopsis wild-type(left) and transgenic plants expressing the 3′-end of the senescence DHSgene (sequence shown in FIG. 36) in antisense orientation showingincreased leaf size in the transgenic plants.

[0075]FIG. 24 is a photograph of 6.1 week old Arabidopsis wild-type(left) and transgenic plants expressing the 3′-end of the senescence DHSgene (sequence shown in FIG. 36) in antisense orientation showingincreased size of transgenic plants.

[0076]FIG. 25 is a graph showing the increase in seed yield from threeT₁ transgenic Arabidopsis plant lines expressing the senescence-inducedDHS gene in antisense orientation. Seed yield is expressed as volume ofseed. SE for n=30 is shown for wild-type plants.

[0077]FIG. 26 is a photograph of transgenic tomato plants expressing the3′-end of the senescence DHS gene (sequence shown in FIG. 36) inantisense orientation (left) and wild-type plants (right) showingincreased leaf size and increased plant size in the transgenic plants.The photograph was taken 18 days after transfer of the plantlets tosoil.

[0078]FIG. 27 is a photograph of transgenic tomato plants expressing the3′-end of the senescence DHS gene (sequence shown in FIG. 36) inantisense orientation (left) and wild-type plants (right) showingincreased leaf size and increased plant size in the transgenic plants.The photograph was taken 32 days after transfer of the plantlets tosoil.

[0079]FIGS. 28 through 35 are photographs of tomato fruit from wild-type(top panels) and transgenic plants expressing the full-length senescenceDHS gene in antisense orientation (bottom panels). Fruit were harvestedat the breaker stage of development and ripened in a growth chamber.Days after harvest are indicated in the upper left corner of each panel.

[0080]FIG. 36 is the nucleotide (top) (SEQ ID NO:30) and derived aminoacid (bottom) sequence of the 3′-end of the Arabidopsissenescence-induced DHS gene used in antisense orientation to totransform plants.

[0081]FIG. 37 is the nucleotide (top) (SEQ ID NO:31) and derived aminoacid (bottom) sequence of the 3′-end of the tomato senescence-inducedDHS gene used in antisense orientation to transform plants.

[0082]FIG. 38 is the nucleotide (top) (SEQ ID NO:26) and derived aminoacid (bottom) sequence of a 600 base pair Arabidopsis senescence-inducedDHS probe used to isolate the full-length Arabidopsis gene.

[0083]FIG. 39 is the nucleotide (top) (SEQ ID NO:27) and derived aminoacid (bottom) sequence of the 483 base pair carnation senescence-inducedDHS probe used to isolate the full-length carnation gene.

DETAILED DESCRIPTION OF THE INVENTION

[0084] Methods and compositions are provided for altering the expressionof senescence-induced DHS gene(s), senescence-induced elF-5A gene(s) orboth in plant cells. Alteration of expression of senescence-induced DHSand senescence-induced elF-5A, either alone or in combination, in plantsresults in delayed onset of senescence and improved resistance toenvironmental stress and pathogens, thus extending the plant shelf-lifeand/or growth period.

[0085] A full length cDNA sequence encoding a tomato DHS gene exhibitingsenescence-induced expression has been isolated by reverse transcriptasemediated polymerase chain reaction (RT-PCR) using RNA isolated fromchill-injured tomato leaves as a template and using the RT-PCR productto screen a chill-injured, sorbitol-treated tomato leaf cDNA library.Polynucleotide probes corresponding to selected regions of the isolatedtomato leaf cDNA sequence as well as the full length tomato leaf cDNAwere used to determine the presence of mRNA encoding the DHS gene inenvironmentally stressed (chilled) tomato leaves, (dehydrated)sorbitol-treated tomato leaves, ripening tomato fruit and senescingtomato blossoms.

[0086] Primers designed from an Arabidopsis DHS genomic clone were usedto generate a polymerase chain reaction (PCR) product using a senescingArabidopsis leaf cDNA library as template. The Arabidopsis nucleotidesequence has 73% nucleotide sequence identity and 81% amino acidsequence identity with the corresponding sequence of thesenescence-induced tomato DHS.

[0087] The senescence-induced tomato DHS gene of the present inventionwas isolated by using RT-PCR. The upstream primer used to isolate thetomato DHS gene is a 24 nucleotide primer: 5′ AG TCT AGA AGG TGC TCG TCCTGA T 3′ (SEQ ID NO. 3); the downstream primer contains 34 nucleotides:5′ G ACT GCA GTC GAC ATC GAT (T)₁₅ 3′ (SEQ ID NO. 4). Using 100 pmol ofthe downstream primer, a first strand of cDNA was isolated usingstandard RT-PCR. The first strand was then used as template in a RT-PCR,using both the upstream and downstream primers. Separation of the RT-PCRproducts on an agarose gel revealed the presence of three distinct bandsranging in size from 1.5 kb to 600 bp. The three fragments weresubcloned into the plasmid vector, pBluescript™ (Stratagene CloningSystems, LaJolla, Calif.) using XbaI and SalI cloning sites present inthe upstream and downstream primers, respectively, and sequenced. Thesequences of the fragments were compared and aligned with sequencespresent in the GeneBank data base. The results showed the 1.5 kb and 1kb fragments to be tomato DHS sequence. The 600 bp fragment also alignedwith human, yeast and Neurospora DHS sequences.

[0088] The 600 bp RT-PCR fragment was used to screen a tomato (cv. MatchF1 hybrid) cDNA library made from RNA obtained from tomato leaves thathad been treated with 2 M sorbitol for six hours to induce dehydration.The cDNA library was constructed using a AZap™ (Stratagene CloningSystems, LaJolla, Calif.) cDNA library kit. Three identical positivefull-length cDNA clones corresponding to the senescence-induced DHS genewere obtained and sequenced. The nucleotide sequence of thesenescence-induced DHS cDNA clone is shown in SEQ ID NO:1. The cDNAclone encodes a 381 amino acid polypeptide (SEQ ID NO: 2) having acalculated molecular mass of 42.1 KDa.

[0089] Based on the expression pattern of the gene in developing andstressed tomato flowers, fruit and leaves, it is involved in senescence.

[0090] The tomato DHS cDNA sequence was aligned with unidentifiedgenomic sequences in the Arabidopsis thaliana genome bank(http://genome-www.stanford.edu/Arabidopsis). The results showedalignment with an unidentified Arabidopsis genomic sequence (AB107060).The alignment information was used to identify an open reading frame inthe Arabidopsis sequence and generate predicted amino acid sequencetherefrom. The resulting nucleotide and amino acid sequences of thealigned Arabidopsis DHS gene are designated as SEQ ID NO. 5 (FIG. 2A)and SEQ ID NO. 6, respectively.

[0091] Two primers based on short regions of the identified ArabidopsisDHS sequence were generated: primer 1, 5′ GGTGGTGTTGAGGMGATC 3′ (SEQ IDNO. 7); and primer 2, 5′ GGTGCACGCCCTGATGAAGC 3′ (SEQ ID NO. 8). AnArabidopsis senescing leaf cDNA library was used as template for the twoprimers in a standard PCR. A 600 bp PCR product was isolated andsequenced and shown to have an identical sequence as that of thecorresponding fragment of the genomic DHS sequence.

[0092] The full-length senescence-induced tomato DHS cDNA clone was alsoused to isolate full-length senescence-induced Arabidopsis and carnationDHS cDNA clones. The Arabidopsis and carnation DHS cDNA clones wereisolated by screening a senescing Arabidopsis leaf cDNA library and asenescencing carnation petal cDNA library, respectively, using thefull-length tomato DHS cDNA clone as probe. cDNA clones obtained fromthe cDNA libraries were then sequenced. The nucleotide sequence of theArabidopsis full-length cDNA clone isolated in this manner has the samesequence as the coding region of the Arabidopsis genomic sequenceidentified as encoding Arabidopsis DHS by alignment with the tomato cDNAsequence. (FIG. 2A, SEQ ID NO: 5). The nucleotide sequence of thefull-length carnation petal senescence-induced DHS clone and derivedamino acid sequence are shown in FIG. 10 (SEQ ID NO:9 and SEQ ID NO:10,respectively).

[0093] Thus, the cDNA sequences of the invention, encoding DHS fromtomato, carnation and Arabidopsis can be used as probe in a similarmanner to isolate DHS genes from other plants, which can then be used toalter senescence in transgenic plants.

[0094] The senescence-induced DHS gene appears to be a member of a DHSgene family. Genomic Southern blot analysis of tomato leaf DNA wascarried out using genomic DNA extracted from a hybrid plant. The DNA wascut with various restriction enzymes that recognize a single site withinthe coding region of the DHS gene or which do not recognize any siteswithin the open reading frame of the DHS gene. A restriction map fortomato DHS is shown in FIG. 4.

[0095] Restriction enzyme digested tomato leaf genomic DNA was probedwith ³²P-dCTP-labeled full length tomato DHS cDNA. Hybridization underhigh stringency conditions revealed hybridization of the full lengthcDNA probe to two to three restriction fragments for each restrictionenzyme digested DNA sample. Of particular note, when tomato leaf genomicDNA was digested with XbaI and EcoRI, which have restriction siteswithin the open reading frame of DHS (FIG. 4), more than two restrictionfragments were detectable in the Southern blot (FIG. 5). Genomic DNAfrom cv Match F1, a hybrid variety, and the homozygous line, UCT5,yielded the same pattern of restriction fragments. These results suggestthat there are two or more isoforms of the DHS gene in tomato plants. Asshown in FIG. 3, the DHS gene is highly conserved across species and soit would be expected that there is a significant amount of conservationbetween isoforms within any species.

[0096] Northern blots of tomato flower total RNA probed with the fulllength tomato cDNA show that the expression of the senescence-inducedDHS gene is significantly induced in tomato blossoms, but expression isbarely detectable in the buds (FIG. 6).

[0097] Northern blot analysis of DHS expression during variousdevelopmental stages of tomato fruit demonstrate that the DHS gene isexpressed at low levels in breaker and pink fruit, whereas DHSexpression in red (ripe) tomato fruit is significantly enhanced (FIG.7).

[0098] Northern blot analyses also demonstrate that thesenescence-induced DHS gene is induced by environmental stressconditions, e.g., dehydration (FIG. 8) and chilling (FIG. 9). Tomatoleaves that had been treated with 2 M sorbitol to induce dehydrationdemonstrate induction of DHS expression in the dehydrated leavescompared to non-treated leaves (FIG. 8). Plants that have been exposedto chilling temperatures and returned to ambient temperature showinduced expression of the senescence-induced DHS gene coincident withthe development of chilling injury symptoms (e.g., leakiness) (FIG. 9).The overall pattern of gene expression in tomato plants and variousplant tissues, e.g., leaves, fruit and flowers, demonstrates that theDHS gene of the invention is involved in the initiation of senescence inthese plants and plant tissues.

[0099] Similar results in terms of induction of DHS gene expression areobserved with the onset of leaf senescence in Arabidopsis and petalsenescence in carnation. Northern blot analyses of Arabidopsis leaftotal RNA isolated from plants of various ages show that the expressionof the senescence-induced DHS gene is not evident in young(five-week-old plants), but begins to appear at about six weeks.Expression of the DHS gene is significantly induced by seven weeks.Northern blot analysis indicates that the Arabidopsis DHS gene issignificantly enhanced as the plant ages. (FIG. 11).

[0100] Northern blot analyses also demonstrate that the DHS gene issimilarly regulated in flowering plants, such as the carnation. (FIG.12) Northern blot analyses of total RNA isolated from petals ofcarnation flowers of various ages show that the expression of carnationDHS is significantly induced in petals from flowers that have symptomsof age-induced senescence such as petal inrolling, which is the firstmorphological manifestation of senescence, but expression is much lowerin tight-bud flowers. Petals from carnation flowers that are justbeginning to open have significantly more DHS expression than flowers inthe tight-bud stage, and petals from flowers that are fully open alsoshow enhanced expression of DHS.

[0101] Thus, it is expected that by substantially repressing or alteringthe expression of the senescence-induced DHS gene in plant tissues,deterioration and spoilage can be delayed, increasing the shelf-life ofperishable fruits, flowers, and vegetables, and plants and their tissuescan be rendered more stress-tolerant and pathogen resistant. This can beachieved by producing transgenic plants in which the DHS cDNA or anoligonucleotide fragment thereof is expressed in the antisenseconfiguration in fruits, flowers, leaves and vegetables, preferablyusing a constitutive promoter such as the CaMV 35S promoter, or using atissue-specific or senescence/stress-inducible promoter.

[0102] Another gene, elF-5A, which is involved in the induction ofsenescence related morphological changes in plants has also beenisolated and sequenced herein and like the DHS, it can be used to altersenescence and senescence-related processes in plants, preferably, byintroduction in antisense orientation into plants. A full-lengthsenescence-induced elF-5A cDNA clone was isolated from each of ripeningtomato fruit, senescing Arabidopsis leaf and senescing carnation flowercDNA libraries. The nucleotide and derived amino acid sequences of eachof the full length clones is shown in FIGS. 13 (tomatosenescence-induced elF-5A), 14 (carnation senescence-induced elF-5A) and15 (Arabidopsis senescence-induced elF-5A). The nucleotide sequence ofeach of these cDNA clones is also shown as SEQ ID NO: 11 (tomato) (FIG.13), SEQ ID NO:13 (carnation) (FIG. 14) and SEQ ID NO:15 (Arabidopsis)(FIG. 15). The derived amino acid sequence of each of the genes is shownas SEQ ID NO:12 (FIG. 13), SEQ ID NO:14 (FIG. 14) and SEQ ID NO:16 (FIG.15), respectively.

[0103] As is the case with the DHS gene sequences described herein, theelF-5A sequence of the present invention can be used to isolate elF-5Agenes from other plants. The isolated elF-5A sequences can be used toalter senescence and senescence-related processes in plants. Isolationof elF-5A sequences from plants can be achieved using art known methods,based on sequences similarities of at least about 70% across species.

[0104] Parallel induction of elF-5A and DHS occurs in plants duringsenescence. Northern blot analyses demonstrate that elF-5A isupregulated in parallel with DHS at the onset of both natural andstress-induced senescence. (FIGS. 16 through 20) For example, Northernblot analyses of total RNA isolated from leaves of Arabidopsis plants atvarious ages demonstrate that from the time leaf senescence is evidentin the plant the expression of elF-5A is induced and expression issignificantly enhanced as senescence progresses. In fruit bearingplants, such as tomato, elF-5A and DHS are upregulated in parallel inred-soft fruit coincident with the onset of fruit softening andspoilage. (FIG. 17) Northern blot analysis also demonstrates that elF-5Aand DHS are upregulated in parallel in plants in response toenvironmental stress, such as drought (FIG. 18) and chilling injury(FIG. 20). Similarly, in flowering plants, elF-5A and DHS areupregulated in parallel in open flowers and expression of both genescontinues to be enhanced through the later stages of flowering.

[0105] The cloned senescence-induced DHS gene, fragment(s) thereof, orcloned senescence-induced elF-5A gene or fragment(s) thereof, orcombinations of elF-5A and DHS sequences, when introduced in reverseorientation (antisense) under control of a constitutive promoter, suchas the fig wart mosaic virus 35S promoter, cauliflower mosaic viruspromoter CaMV35S, double 35S promoter or MAS promoter, can be used togenetically modify plants and alter senescence in the modified plants.Selected antisense sequences from other plants which share sufficientsequence identity with the tomato, Arabidopsis or carnationsenescence-induced DHS genes or senscence-induced elF-5A genes can beused to achieve similar genetic modification. One result of the geneticmodification is a reduction in the amount of endogenous translatablesenescence-induced DHS-encoding mRNA, elF-5A-encoding mRNA or both.Consequently, the amount of senescence-induced DHS and/orsenescence-induced elF-5A produced in the plant cells is reduced,thereby reducing the amount of activated elF-5A, which in turn reducestranslation of senescence induced proteins, including senescence-inducedlipase, senescence-induced proteases and senescence-induced nucleases.Senescence is thus inhibited or delayed, since de novo protein synthesisis required for the onset of senescence.

[0106] For example, Arabidopsis plants transformed with vectors thatexpress either the full-length or 3′-region of the Arabidopsissenescence-induced DHS gene (SEQ ID NO:26) (FIG. 38) in antisenseorientation, under regulation of a double 35S promoter exhibit increasedbiomass, e.g., larger leaf size and overall larger plant growththroughout all stages of growth, and delayed leaf senescence incomparison to control plants as shown in FIGS. 21 through 24.

[0107] The effect of reduced expression of the senescence-induced DHSgene brought about by expressing either the full-length or 3′ codingregion of the Arabidopsis senescence-induced DHS gene in antisenseorientation in transgenic Arabidopsis plants is also seen as an increasein seed yield in the transformed plants. Arabidopsis plant linesexpressing the antisense 3′ non-coding region of the Arabidopsissenescence-induced DHS gene produce up to six times more seed than wildtype plants. (FIG. 25) Similar results are obtained with tomato plantstransformed with the 3′ end of the tomato senescence-induced DHS gene(SEQ ID NO:27) in antisense orientation and under regulation of a double35S promoter. Plants transformed with the 3′ end of the gene inantisense orientation show increased leaf size and increased plant sizein comparison to control (non-transformed) tomato plants. (FIGS. 26 and27)

[0108] Tomato plants transformed with the full length tomatosenescence-induced DHS in antisense orientation produce fruit thatexhibits delayed softening and spoilage in comparison to wild typeplants. (FIGS. 28 through 35). Thus, the methods and sequences of thepresent invention can be used to delay fruit softening and spoilage, aswell as to increase plant biomass and seed yield and in general, delaysenesence in plants.

[0109] The isolated nucleotide sequences of this invention can be usedto isolate substantially complementary DHS and'or elF-5A nucleotidesequence from other plants or organisms. These sequences can, in turn,be used to transform plants and thereby alter senescence of thetransformed plants in the same manner as shown with the use of theisolated nucleotide sequences shown herein.

[0110] The genetic modifications obtained with transformation of plantswith DHS, elF-5A, fragments thereof or combinations thereof can effect apermanent change in levels of senescence-induced DHS, elF-5A or both inthe plant and be propagated in offspring plants by selfing or otherreproductive schemes. The genetically altered plant is used to produce anew variety or line of plants wherein the alteration is stablytransmitted from generation to generation. The present inventionprovides for the first time the appropriate DNA sequences which may beused to achieve a stable genetic modification of senescence in a widerange of different plants.

[0111] For the identification and isolation of the senescence-inducedDHS gene and elF-5A gene, in general, preparation of plasmid DNA,restriction enzyme digestion, agarose gel electrophoresis of DNA,polyacrylamide gel electrophoresis of protein, PCR, RT-PCR, Southernblots, Northern blots, DNA ligation and bacterial transformation werecarried out using conventional methods well-known in the art. See, forexample, Sambrook, J. et al., Molecular Cloning: A Laboratory Manual,2nd ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989.Techniques of nucleic acid hybridization are disclosed by Sambrook(Supra).

[0112] As used herein, the term “plant” refers to either a whole plant,a plant part, a plant cell or a group of plant cells. The type of plantwhich can be used in the methods of the invention is not limited andincludes, for example, ethylene-sensitive and ethylene-insensitiveplants; fruit bearing plants such as apricots, apples, oranges, bananas,grapefruit, pears, tomatoes, strawberries, avocados, etc.; vegetablessuch as carrots, peas, lettuce, cabbage, turnips, potatoes, broccoli,asparagus, etc.; flowers such as carnations, roses, mums, etc.;agronomic crop plants and forest species such as corn, rice, soybean,alfalfa and the like; and in general, any plant that can take up andexpress the DNA molecules of the present invention. It may includeplants of a variety of ploidy levels, including haploid, diploid,tetraploid and polyploid. The plant may be either a monocotyledon ordicotyledon.

[0113] A transgenic plant is defined herein as a plant which isgenetically modified in some way, including but not limited to a plantwhich has incorporated heterologous or homologous senescence-induced DHSDNA or modified DNA or some portion of heterologous senescence-inducedDHS DNA or homologous senescence-induced DHS DNA into its genome.Alternatively a transgenic plant of the invention may have incorporatedheterologous or homologous senescence-induced elF-5A DNA or modified DNAor some portion of heterologous senescence-induced elF-5A DNA orhomologous senescence-induced elF-5A DNA into its genome. Transgenicplants of the invention may have incorporated heterologous or homologoussenescence-induced DHS and elF-5A DNA or modified DNA or some portion ofheterologous senescence-induced DHS and elF-5A DNA or homologoussenescence-induced DHS DNA or a combination of heterologous andhomologous DHS and elF-5A sequences into its genome. The altered geneticmaterial may encode a protein, comprise a regulatory or controlsequence, or may be or include an antisense sequence or encode anantisense RNA which is antisense to the endogenous senescence-inducedDHS or elF-5A DNA or mRNA sequence or portion thereof of the plant. A“transgene” or “transgenic sequence” is defined as a foreign gene orpartial sequence which has been incorporated into a transgenic plant.

[0114] The term “hybridization” as used herein is generally used to meanhybridization of nucleic acids at appropriate conditions of stringencyas would be readily evident to those skilled in the art depending uponthe nature of the probe sequence and target sequences. Conditions ofhybridization and washing are well known in the art, and the adjustmentof conditions depending upon the desired stringency by varyingincubation time, temperature and/or ionic strength of the solution arereadily accomplished. See, for example, Sambrook, J. et al., MolecularCloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Press,Cold Spring Harbor, N.Y., 1989. The choice of conditions is dictated bythe length of the sequences being hybridized, in particular, the lengthof the probe sequence, the relative G-C content of the nucleic acids andthe amount of mismatches to be permitted. Low stringency conditions arepreferred when partial hybridization between strands that have lesserdegrees of complementarity is desired. When perfect or near perfectcomplementarity is desired, high stringency conditions are preferred.For typical high stringency conditions, the hybridization solutioncontains 6×S.S.C., 0.01 M EDTA, 1× Denhardt's solution and 0.5% SDS.Hybridization is carried out at about 68° C. for about 3 to 4 hours forfragments of cloned DNA and for about 12 to about 16 hours for totaleukaryotic DNA. For lower stringencies the temperature of hybridizationis reduced to about 42° C. below the melting temperature (TM) of theduplex. The TM is known to be a function of the G-C content and duplexlength as well as the ionic strength of the solution.

[0115] As used herein, the term “substantial sequence identity” or“substantial homology” is used to indicate that a nucleotide sequence oran amino acid sequence exhibits substantial structural or functionalequivalence with another nucleotide or amino acid sequence. Anystructural or functional differences between sequences havingsubstantial sequence identity or substantial homology will be deminimis; that is, they will not affect the ability of the sequence tofunction as indicated in the desired application. Differences may be dueto inherent variations in codon usage among different species, forexample. Structural differences are considered de minimis if there is asignificant amount of sequence overlap or similarity between two or moredifferent sequences or if the different sequences exhibit similarphysical characteristics even if the sequences differ in length orstructure. Such characteristics include, for example, ability tohybridize under defined conditions, or in the case of proteins,immunological crossreactivity, similar enzymatic activity, etc. Each ofthese characteristics can readily be determined by the skilledpractitioner by art known methods.

[0116] Additionally, two nucleotide sequences are “substantiallycomplementary” if the sequences have at least about 70 percent, morepreferably, 80 percent and most preferably about 90 percent sequencesimilarity between them. Two amino acid sequences are substantiallyhomologous if they have at least 50%, preferably 70% similarity betweenthe active portions of the polypeptides.

[0117] As used herein, the phrase “hybridizes to a correspondingportion” of a DNA or RNA molecule means that the molecule thathybridizes, e.g., oligonucleotide, polynucleotide, or any nucleotidesequence (in sense or antisense orientation) recognizes and hybridizesto a sequence in another nucleic acid molecule that is of approximatelythe same size and has enough sequence similarity thereto to effecthybridization under appropriate conditions. For example, a 100nucleotide long antisense molecule from the 3′ coding or non-codingregion of tomato DHS will recognize and hybridize to an approximately100 nucleotide portion of a nucleotide sequence within the 3′ coding ornon-coding region, respectively of carnation DHS gene or any other plantDHS gene so long as there is about 70% or more sequence similaritybetween the two sequences. It is to be understood that the size of the“corresponding portion” will allow for some mismatches in hybridizationsuch that the “corresponding portion” may be smaller or larger than themolecule which hybridizes to it, for example 20-30% larger or smaller,preferably no more than about 12-15% larger or smaller.

[0118] The term “functional derivative” of a nucleic acid (or poly- oroligonucleotide) is used herein to mean a fragment, variant, homolog, oranalog of the gene or nucleotide sequence encoding senescence-inducedDHS or senescence-induced elF-5A. A functional derivative may retain atleast a portion of the function of the senescence-induced DHS or elF-5Aencoding DNA which permits its utility in accordance with the invention.Such function may include the ability to hybridize under low stringencyconditions with native tomato, Arabidopsis or carnationsenescence-induced DHS or elF-5A or substantially homologous DNA fromanother plant which encodes senescence-induced DHS or elF-5A or with anmRNA transcript thereof, or, in antisense orientation, to inhibit thetranscription and/or translation of plant senescence-induced DHS orelF-5A mRNA, or the like.

[0119] A “fragment” of the gene or DNA sequence refers to any subset ofthe molecule, e.g., a shorter polynucleotide or oligonucleotide. A“variant” refers to a molecule substantially similar to either theentire gene or a fragment thereof, such as a nucleotide substitutionvariant having one or more substituted nucleotides, but which maintainsthe ability to hybridize with the particular gene or to encode mRNAtranscript which hybridizes with the native DNA. A “homolog” refers to afragment or variant sequence from a different plant genus or species. An“analog” refers to a non-natural molecule substantially similar to orfunctioning in relation to either the entire molecule, a variant or afragment thereof.

[0120] By “altered expression” or “modified expression” of a gene, e.g.,the senescence-induced DHS gene or senescence-induced elF-5A gene, ismeant any process or result whereby the normal expression of the gene,for example, that expression occurring in an unmodified fruit bearing,flowering or other plant, is changed in some way. As intended herein,alteration in gene expression is complete or partial reduction in theexpression of the senescence-induced DHS gene or senescence-inducedelF-5A gene or both, but may also include a change in the timing ofexpression, or another state wherein the expression of thesenescence-induced DHS gene or senescence-induced elF-5A gene or bothdiffers from that which would be most likely to occur naturally in anunmodified plant or cultivar. A preferred alteration is one whichresults in reduction of senescence-induced DHS production,senescence-induced elF-5A production or both by the plant compared toproduction in an unmodified plant.

[0121] In producing a genetically altered plant in accordance with thisinvention, it is preferred to select individual plantlets or plants bythe desired trait, generally reduced senescence-induced DHS expressionor production or reduced senescence-induced elF-5A expression or both.Expression of senescence-induced DHS and senescence-induced elF-5A canbe determined, for example by observations of delayed or reducedsenescence in transgenic plants. It is also possible to quantitate theactivity of DHS and/or elF-5A in transgenic plants in comparison tocontrol (normal, non-transgenic) plants using known assays.

[0122] In order for a newly inserted gene or DNA sequence to beexpressed, resulting in production of the protein which it encodes, orin the case of antisense DNA, to be transcribed, resulting in anantisense RNA molecule, the proper regulatory elements should be presentin proper location and orientation with respect to the gene or DNAsequence. The regulatory regions may include a promoter, a5′-non-translated leader sequence and a 3′-polyadenylation sequence aswell as enhancers and other regulatory sequences.

[0123] Promoter regulatory elements that are useful in combination withthe senescence-induced DHS gene to generate sense or antisensetranscripts of the gene include any plant promoter in general, and moreparticularly, a constitutive promoter such as the fig wart mosaic virus35S promoter, the cauliflower mosaic virus promoter, CaMV35S promoter,or the MAS promoter, or a tissue-specific or senescence-inducedpromoter, such as the carnation petal GST1 promoter or the ArabidopsisSAG12 promoter (See, for example, J. C. Palaqui et al., Plant Physiol.,112:1447-1456 (1996); Morton et al., Molecular Breeding, 1:123-132(1995); Fobert et al., Plant Journal, 6:567-577 (1994); and Gan et al.,Plant Physiol., 113:313 (1997), incorporated herein by reference).Preferably, the promoter used in the present invention is a constitutivepromoter, most preferably a double 35S promoter is used.

[0124] Expression levels from a promoter which is useful for the presentinvention can be tested using conventional expression systems, forexample by measuring levels of a reporter gene product, e.g., protein ormRNA in extracts of the leaves, flowers, fruit or other tissues of atransgenic plant into which the promoter/reporter gene have beenintroduced.

[0125] The present invention provides antisense oligonucleotides andpolynucleotides complementary to the gene encoding tomatosenescence-induced DHS, carnation senescence-induced DHS, Arabidopsissenescence-induced DHS or complementary to a gene or gene fragment fromanother plant, which hybridizes with the tomato, carnation orArabidopsis senescence-induced DHS gene under low to high stringencyconditions. The present invention also provides antisenseoligonucleotides and polynucleotides complementary to the gene encodingtomato senescence-induced elF-5A, carnation senescence-induced elF-5A,Arabidopsis senescence-induced elF-5A or complementary to a gene or genefragment from another plant, which hybridizes with the tomato, carnationor Arabidopsis senescence-induced elF-5A gene under low to highstringency conditions. Such antisense oligonucleotides should be atleast about six nucleotides in length to provide minimal specificity ofhybridization and may be complementary to one strand of DNA or mRNAencoding the senescence-induced gene or a portion thereof, or toflanking sequences in genomic DNA which are involved in regulatingsenescence-induced DHS or elF-5A gene expression. The antisenseoligonucleotide may be as large as 100 nucleotides or more and mayextend in length up to and beyond the full coding sequence for which itis antisense. The antisense oligonucleotides can be DNA or RNA orchimeric mixtures or derivatives or modified versions thereof, singlestranded or double stranded.

[0126] The action of the antisense oligonucleotide may result inalteration, primarily inhibition, of senescence-induced DHS expression,senescence-induced elF-5A expression or both in cells. For a generaldiscussion of antisense see: Alberts, et al., Molecular Biology of theCell, 2nd ed., Garland Publishing, Inc. New York, N.Y., 1989 (inparticular pages 195-196, incorporated herein by reference).

[0127] The antisense oligonucleotide may be complementary to anycorresponding portion of the senescence-induced DHS or elF-5A gene. Inone embodiment, the antisense oligonucleotide may be between 6 and 100nucleotides in length, and may be complementary to the 5′-non-coding orsequences within the 3′-end of the senescence-induced DHS or elF-5Asequence, for example. Antisense oligonucleotides primarilycomplementary to 5′-non-coding sequences are known to be effectiveinhibitors of expression of genes encoding transcription factors.Branch, M. A., Molec. Cell Biol., 13:4284-4290 (1993).

[0128] Preferred antisense oligonucleotides are substantiallycomplementary to a portion of the mRNA encoding senescence-induced DHSor senescence-induced elF-5A, the portion of the mRNA beingapproximately the same size as the antisense oligonuleotide. Forexample, introduction of the full length cDNA clone encodingsenescence-induced DHS or elF-5A in an antisense orientation into aplant is expected to result in successfully altered senescence-inducedDHS and/or elF-5A gene expression. Moreover, as demonstrated in FIGS.21-35 introduction of partial sequences, targeted to specific portionsof the senescence-induced DHS gene or senescence-induced elF-5A gene orboth, can be equally effective.

[0129] The minimal amount of homology required by the present inventionis that sufficient to result in sufficient complementarity to providerecognition of the specific target RNA or DNA and inhibition orreduction of its translation or function while not affecting function ofother RNA or DNA molecules and the expression of other genes. While theantisense oligonucleotides of the invention comprise sequencescomplementary to a corresponding portion of an RNA transcript of thesenescence-induced DHS gene or senescence-induced elF-5A gene, absolutecomplementarity, although preferred is not required. The ability tohybridize may depend on the length of the antisense oligonucleotide andthe degree of complementarity. Generally, the longer the hybridizingnucleic acid, the more base mismatches with the senescence-induced DHStarget sequence it may contain and still form a stable duplex. Oneskilled in the art can ascertain a tolerable degree of mismatch by useof standard procedures to determine the melting temperature of thehybridized complex, for example.

[0130] The antisense RNA oligonucleotides may be generatedintracellularly by transcription from exogenously introduced nucleicacid sequences. The antisense molecule may be delivered to a cell bytransformation or transfection or infection with a vector, such as aplasmid or virus into which is incorporated DNA encoding the antisensesenescence-induced DHS sequence operably linked to appropriateregulatory elements, including a promoter. Within the cell the exogenousDNA sequence is expressed, producing an antisense RNA of thesenescence-induced DHS gene.

[0131] Vectors can be plasmids, preferably, or may be viral or othervectors known in the art to replicate and express genes encoded thereonin plant cells or bacterial cells. The vector becomes chromosomallyintegrated such that it can be transcribed to produce the desiredantisense senescence-induced DHS RNA. Such plasmid or viral vectors canbe constructed by recombinant DNA technology methods that are standardin the art. For example, the vector may be a plasmid vector containing areplication system functional in a prokaryotic host and an antisenseoligonucleotide or polynucleotide according to the invention.Alternatively, the vector may be a plasmid containing a replicationsystem functional in Agrobacterium and an antisense oligonucleotide orpolynucleotide according to the invention. Plasmids that are capable ofreplicating in Agrobacterium are well known in the art. See, Miki, etal., Procedures for Introducing Foreign DNA Into Plants, Methods inPlant Molecular Biology and Biotechnology, Eds. B. R. Glick and J. E.Thompson. CRC Press (1993), PP. 67-83.

[0132] The tomato DHS gene was cloned in antisense orientation into aplasmid vector in the following manner. The pCD plasmid, which isconstructed from a pUC18 backbone and contains the 35S promoter fromcauliflower mosaic virus (CaMV) followed by a multiple cloning site andan octapine synthase termination sequence was used for cloning thetomato DHS gene. The pCd-DHS (antisense) plasmid was constructed bysubcloning the full length tomato DHS gene in the antisense orientationinto the pCD plasmid using XhoI and SacI restriction sites.

[0133] An oligonucleotide, preferably between about 6 and about 100nucleotides in length and complementary to the target sequence ofsenescence-induced DHS or senescence-induced elF-5A gene, may beprepared by recombinant nucleotide technologies or may be synthesizedfrom mononucleotides or shorter oligonucleotides, for example. Automatedsynthesizers are applicable to chemical synthesis of the oligo- andpolynucleotides of the invention. Procedures for constructingrecombinant nucleotide molecules in accordance with the presentinvention are disclosed in Sambrook, et al., In: Molecular Cloning: ALaboratory Manual, 2nd ed., Cold Spring Harbor Press, Cold SpringHarbor, N.Y. (1989), which is incorporated herein in its entirety.Oligonucleotides which encode antisense RNA complementary tosenescence-induced deoxyhypusine synthase sequence can be prepared usingprocedures well known to those in the art. Details concerning suchprocedures are provided in Maniatis, T. et al., Molecular mechanisms inthe Control of Gene expression, eds., Nierlich, et al., eds., Acad.Press, N.Y. (1976).

[0134] In an alternative embodiment of the invention, inhibition ofexpression of endogenous plant senescence-induced DHS,senescence-induced elF-5A or both is the result of co-suppressionthrough over-expression of an exogenous senescence-induced DHS or elF-5Agene or gene fragment or both introduced into the plant cell. In thisembodiment of the invention, a vector encoding senescence-induced DHS,senescence-induced elF-5A or both in the sense orientation is introducedinto the cells in the same manner as described herein for antisensemolecules. Preferably, the senescence-induced DHS or senescence-inducedelF-5A is operatively linked to a strong constitutive promoter, such asfor example the fig wart mosaic virus promoter or CaMV35S or a double 35S promoter.

[0135] In another embodiment of the invention, inhibition of expressionof endogenous plant senescence-induced DHS, senescence-induced elF-5A orboth is effected through the use of ribozymes. Ribozymes are RNAmolecules exhibiting sequence-specific endoribonuclease activity. Anexample is the hammerhead ribozyme which cleaves at a UH (where H is anA, C or U residue) recognition site in the target RNA and containsbase-pairing regions that direct the catalytic domain of the ribozyme tothe target site of the substrate RNA. Ribozymes are highlytarget-specific and can be designed to inactivate one member of amultigene family or targeted to conserved regions of related mRNAs. (SeeMerlo et al., The Plant Cell, 10:1603-1621, 1998). The ribozyme moleculemay be delivered to a cell by transformation, transfection or infectionwith a vector, such as a plasmid or virus, into which is incorporatedthe ribozyme operatively linked to appropriate regulatory elements,including a promoter. Such a ribozyme construct contains base-pairingarms that direct it to a cleavage site within the senescence-induced DHSmRNA, or senescence-induced elF-5A mRNA resulting in cleavage of DHS orelF-5A mRNA and inhibition of senescence-induced DHS and/or elF-5Aexpression.

[0136] Transgenic plants made in accordance with the present inventionmay be prepared by DNA transformation using any method of planttransformation known in the art. Plant transformation methods includedirect co-cultivation of plants, tissues or cells with Agrobacteriumtumefaciens or direct infection (Miki, et al., Meth. in Plant Mol. Biol.and Biotechnology, (1993), p. 67-88); direct gene transfer intoprotoplasts or protoplast uptake (Paszkowski, et al., EMBO J., 12:2717(1984); electroporation (Fromm, et al., Nature, 319:719 (1986); particlebombardment (Klein et al., BioTechnology, 6:559-563 (1988); injectioninto meristematic tissues of seedlings and plants (De LaPena, et al.,Nature, 325:274-276 (1987); injection into protoplasts of cultured cellsand tissues (Reich, et al., BioTechnology, 4:1001-1004 (1986)).

[0137] Generally a complete plant is obtained from the transformationprocess. Plants are regenerated from protoplasts, callus, tissue partsor explants, etc. Plant parts obtained from the regenerated plants inwhich the expression of senescence-induced DHS, senescence-inducedelF-5A or both is altered, such as leaves, flowers, fruit, seeds and thelike are included in the definition of “plant” as used herein. Progeny,variants and mutants of the regenerated plants are also included in thedefinition of “plant.”

[0138] The tomato, carnation or Arabidopsis senescence-induced DHSprotein or functional derivatives thereof, and tomato, carnation orArabidopsis senescence-induced elF-5A protein or functional derivativesthereof are preferably produced by recombinant technologies, optionallyin combination with chemical synthesis methods. In one embodiment of theinvention the senescence-induced DHS is expressed as a fusion protein,preferably consisting of the senescence-induced DHS fused with maltosebinding protein.

[0139] “Functional derivatives” of the senescence-induced DHS orsenescence-induced elF-5A protein as described herein are fragments,variants, analogs, or chemical derivatives of senescence-induced DHS orsenescence-induced elF-5A, respectively, which retain at least a portionof the senescence-induced DHS or elF-5A activity or immunological crossreactivity with an antibody specific for senescence-induced DHS orsenescence-induced elF-5A, respectively. A fragment of thesenescence-induced DHS or senescence-induced elF-5A protein refers toany subset of the molecule. Variant peptides may be made by directchemical synthesis, for example, using methods well known in the art. Ananalog of senescence-induced DHS or senescence-induced elF-5A refers toa non-natural protein substantially similar to either the entire proteinor a fragment thereof. Chemical derivatives of senescence-induced DHS orsenescence-induced-elF-5A contain additional chemical moieties notnormally a part of the peptide or peptide fragment. Modifications may beintroduced into peptides or fragments thereof by reacting targeted aminoacid residues of the peptide with an organic derivatizing agent that iscapable of reacting with selected side chains or terminal residues.

[0140] A senescence-induced DHS or senescence-induced elF-5A protein orpeptide according to the invention may be produced by culturing a celltransformed with a nucleotide sequence of this invention (in the senseorientation), allowing the cell to synthesize the protein and thenisolating the protein, either as a free protein or as a fusion protein,depending on the cloning protocol used, from either the culture mediumor from cell extracts. Alternatively, the protein can be produced in acell-free system. Ranu, et al., Meth. Enzymol., 60:459-484, (1979).

[0141] Having now generally described the invention, the same will bemore readily understood through reference to the following exampleswhich are provided by way of illustration, and are not intended to belimiting to the present invention.

EXAMPLE 1

[0142] Messenger RNA (mRNA) Isolation

[0143] Total RNA was isolated from tomato flowers and tomato fruit atvarious developmental stages and from leaves (untreated or afterchilling or sorbitol treatment). Briefly, the tissue (5 g) was ground inliquid nitrogen. The ground powder was mixed with 30 ml guanidiniumbuffer (4 M guanidinium isothiocyanate, 2.5 mM NaOAc pH 8.5, 0.8%β-mercaptoethanol). The mixture was filtered through four layers ofcheesecloth and centrifuged at 10,000×g at 4° C. for 30 minutes. Thesupernatant was then subjected to cesium chloride density gradientcentrifugation at 26,000×g for 20 hours. The pelleted RNA was rinsedwith 75% ethanol, resuspended in 600 μl DEPC-treated water and the RNAprecipitated at −70° C. with 0.75 ml 95% ethanol and 30 μl of 3M NaOAc.Ten μg of RNA were fractionated on a 1.2% denaturing formaldehydeagarose gel and transferred to a nylon membrane. Randomly primed³²P-dCTP-labelled full length DHS cDNA (SEQ ID NO:1) was used to probethe membrane at 42° C. overnight. The membrane was then washed once in1×SSC containing 0.1% SDS at room temperature for 15 minutes and threetimes in 0.2×SSC containing 0.1% SDS at 65° C. for 15 minutes each. Themembrane was exposed to x-ray film overnight at −70° C.

[0144] PolyA⁺ mRNA was isolated from total RNA using the PolyA⁺ tractmRNA Isolation System available from Promega. PolyA⁺ mRNA was used as atemplate for cDNA synthesis using the ZAP Express® cDNA synthesis systemavailable from Stratagene (La Jolla, Calif.)

[0145] Tomato Leaf cDNA Library Screening

[0146] A cDNA library made using mRNA isolated from Match Fl hybridtomato leaves that had been exposed to 2 M sorbitol for six hours wasdiluted to approximately 5×10⁶ PFU/ml. The cDNA library was screenedusing a ³²P-labelled 600 bp RT-PCR fragment. Three positive cDNA cloneswere excised and recircularized into a pBK-CMV® (Stratagene) phagemidusing the method in the manufacturer's instructions. The full lengthcDNA was inserted into the pBK-CMV vector.

[0147] Plasmid DNA Isolation, DNA Sequencing

[0148] The alkaline lysis method described by Sambrook et al., (Supra)was used to isolate plasmid DNA. The full length positive cDNA clone wassequenced using the dideoxy sequencing method. Sanger, et al., Proc.Natl. Acad. Sci. USA, 74:5463-5467. The open reading frame was compiledand analyzed using BLAST search (GenBank, Bethesda, Md.) and alignmentof the five most homologous proteins with the derived amino acidsequence of the encoded gene was achieved using a BCM Search Launcher:Multiple Sequence Alignments Pattern-Induced Multiple Alignment Method(See F. Corpet, Nuc. Acids Res., 16:10881-10890, (1987)). Functionalmotifs present in the derived amino acid sequence were identified byMultiFinder.

[0149] Northern Blot Hybridizations of Tomato RNA

[0150] Ten μg of total RNA isolated from tomato flowers at variousstages (bud and blossom and senescing petals that are open widely ordrying), tomato leaves, and tomato fruit at various stages of ripening(breaker, i.e., green fruit with less than 10% red color, pink, i.e.,the entire fruit is orange or pink, and red, either soft or firm) wereseparated on 1% denatured formaldehyde agarose gels and immobilized onnylon membranes. The full length tomato cDNA labelled with ³²P-dCTPusing a random primer kit (Boehringer Mannheim) was used to probe thefilters (7×10⁷ cpm). The filters were washed once with 1×SSC, 0.1% SDSat room temperature and three times with 0.2×SSC, 0.1% SDS at 65° C. Thefilters were dried and exposed to X-ray film overnight at −70° C. Theresults are shown in FIGS. 6, 7, 8 and 9.

[0151] Northern Blot Hybridization of Arabidopsis RNA

[0152] Total RNA from leaves of Arabidopsis plants at five weeks of age(lane 1), six weeks (lane 2) and seven weeks (lane 3) was isolated asabove, separated on 1% denatured formaldehyde agarose gels andimmobilized on nylon membranes. The full-length Arabidopsissenescence-induced DHS cDNA labelled with ³²P-dCTP using a random primerkit (Boehringer Mannheim) was used to probe the filters (7×10⁷ cpm). Thefilters were washed once with 1×SSC, 0.1% SDS at room temperature andthree times with 0.2×SSC, 0.1% SDS at 65° C. The filters were dried andexposed to X-ray film overnight at −70° C. The results are shown in FIG.11.

[0153] Northern Blot Hybridization of Carnation RNA

[0154] Total RNA from petals of carnation plants at various stages offlower development, i.e., tight-bud flowers (lane 1), beginning to open(lane 2), fully open flowers (lane 3), flowers with inrolling petals(lane 4), was isolated as above, separated on 1% denatured formaldehydeagarose gels and immobilized on nylon membranes. The full-lengthcarnation senescence-induced DHS cDNA labelled with ³²P-dCTP using arandom primer kit (Boehringer Mannheim) was used to probe the filters(7×10⁷ cpm). The filters were washed once with 1×SSC, 0.1% SDS at roomtemperature and three times with 0.2×SSC, 0.1% SDS at 65° C. The filterswere dried and exposed to X-ray film overnight at −70° C. The resultsare shown in FIG. 12.

EXAMPLE 2

[0155] Sorbitol Induction of Tomato Senescence-Induced DHS Gene

[0156] Tomato leaves were treated with 2 M sorbitol in a sealed chamberfor six hours. RNA was extracted from the sorbitol treated leaves asfollows.

[0157] Leaves (5 g) were ground in liquid nitrogen. The ground powderwas mixed with 30 ml guanidinium buffer (4 M guanidinium isothiocyanate,2.5 mM NaOAc pH 8.5, 0.8% β-mercaptoethanol). The mixture was filteredthrough four layers of cheesecloth and centrifuged at 10,000×g at 4° C.for 30 minutes. The supernatant was then subjected to cesium chloridedensity gradient centrifugation at 26,000×g for 20 hours. The pelletedRNA was rinsed with 75% ethanol, resuspended in 600 μl DEPC-treatedwater and the RNA precipitated at −70° C. with 0.75 ml 95% ethanol and30 μl of 3M NaOAc. Ten μg of RNA were fractionated on a 1.2% denaturingformaldehyde agarose gel and transferred to a nylon membrane. Randomlyprimed ³²P-dCTP-labelled full length DHS cDNA (SEQ ID NO:1) was used toprobe the membrane at 42° C. overnight. The membrane was then washedonce in 1×SSC containing 0.1% SDS at room temperature for 15 minutes andthree times in 0.2×SSC containing 0.1% SDS at 65° C. for 15 minuteseach. The membrane was exposed to x-ray film overnight at −70° C.

[0158] The results are shown in FIG. 8. As can be seen, transcription ofDHS is induced in leaves by sorbitol.

EXAMPLE 3

[0159] Induction of the Tomato DHS Gene in Senescing Flowers

[0160] Tight flower buds and open, senescing flowers of tomato plantswere harvested, and RNA was isolated as in Example 2. Ten μg RNA werefractionated on a 1.2% denaturing formaldehyde agarose gel andtransferred to a nylon membrane. Randomly primed ³²P-dCTP-labelled fulllength DHS cDNA (SEQ ID NO.1) was used to probe the membrane at 42° C.overnight. The membrane then was washed once in 1×SSC containing 0.1%SDS at room temperature for 15 minutes and then washed three times in0.2×SSC containing 0.1% SDS at 65° C. for fifteen minutes each. Themembrane was exposed to x-ray film overnight at −70° C.

[0161] The results are shown in FIG. 6. As can be seen, transcription ofDHS is induced in senescing flowers.

EXAMPLE 4

[0162] Induction of the Tomato DHS Gene in Ripening Fruit

[0163] RNA was isolated from breaker, pink and ripe fruit as in Example2. Ten μg RNA were fractionated on a 1.2% denaturing formaldehydeagarose gel and transferred to a nylon membrane. Randomly primed³²P-dCTP-labelled full length DHS cDNA (SEQ ID NO.1) (FIG. 1) was usedto probe the membrane at 42° C. overnight. The membrane then was washedonce in 1×SSC containing 0.1% SDS at room temperature for 15 minutes andthen washed three times in 0.2×SSC containing 0.1% SDS at 65° C. forfifteen minutes each. The membrane was exposed to x-ray film overnightat −70° C.

[0164] The results are shown in FIG. 7. As can be seen, transcription ofDHS is strongest in ripe, red fruit just prior to the onset ofsenescence leading to spoilage.

EXAMPLE 5

[0165] Induction of Tomato Senescence-Induced DHS Gene by Chilling

[0166] Tomato plants in pots (7-8 weeks old) were exposed to 6° C. fortwo days, three days or six days in a growth chamber. The light cyclewas set for eight hours of dark and sixteen hours of light. Plants wererewarmed by moving them back into a greenhouse. Plants that were notrewarmed were harvested immediately after removal from the growthchamber. RNA was extracted from the leaves as follows.

[0167] Leaves (5 g) were ground in liquid nitrogen. The ground powderwas mixed with 30 ml guanidinium buffer (4 M guanidinium isothiocyanate,2.5 mM NaOAc pH 8.5, 0.8% β-mercaptoethanol). The mixture was filteredthrough four layers of cheesecloth and centrifuged at 10,000 g at 4° C.for 30 minutes. The supernatant was then subjected to cesium chloridedensity gradient centrifugation at 26,000 g for 20 hours. The pelletedRNA was rinsed with 75% ethanol, resuspended in 600 μl DEPC-treatedwater and the RNA precipitated at −70° C. with 0.75 ml 95% ethanol and30 μl of 3M NaOAc. Ten μg of RNA were fractionated on a 1.2% denaturingformaldehyde agarose gel and transferred to a nylon membrane. Randomlyprimed ³²P-dCTP-labelled full length DHS cDNA (SEQ ID NO:1) was used toprobe the membrane at 42° C. overnight. The membrane was then washedonce in 1×SSC containing 0.1% SDS at room temperature for 15 minutes andthree times in 0.2×SSC containing 0.1% SDS at 65° C. for 15 minuteseach. The membrane was exposed to x-ray film overnight at −70° C.

[0168] The results are shown in FIG. 9. As can be seen, transcription ofDHS is induced in leaves by exposure to chilling temperature andsubsequent rewarming, and the enhanced transcription correlates withchilling damage measured as membrane leakiness.

EXAMPLE 6

[0169] Generation of an Arabidopsis PCR Product Using Primers Based onUnidentified Arabidopsis Genomic Sequence

[0170] A partial length senescence-induced DHS sequence from anArabidopsis cDNA template was generated by PCR using a pair ofoligonucleotide primers designed from Arabidopsis genomic sequence. The5′ primer is a 19-mer having the sequence, 5′-GGTGGTGTTGAGGAAGATC (SEQID NO:7); the 3′ primer is a 20 mer having the sequence,GGTGCACGCCCTGATGAAGC-3′ (SEQ ID NO:8). A polymerase chain reaction usingthe Expand High Fidelity PCR System (Boehringer Mannheim) and anArabidopsis senescing leaf cDNA library as template was carried out asfollows. Reaction components: cDNA   1 μl (5 × 10⁷ pfu) dNTP (10 mMeach)   1 μl MgCl₂ (5 mM) + 10 × buffer   5 μl Primers 1 and 2 (100 μMeach)   2 μl Expand High Fidelity DNA polymerase 1.75 U Reaction volume  50 μl Reaction paramaters: 94° C. for 3 min 94° C./1 min, 58° C./1min, 72° C./2 min, for 45 cycles 72° C. for 15 min.

EXAMPLE 7

[0171] Isolation of Genomic DNA and Southern Analysis

[0172] Genomic DNA was extracted from tomato leaves by grinding 10 gramsof tomato leaf tissue to a fine powder in liquid nitrogen. 37.5 ml of amixture containing 25 ml homogenization buffer [100 mM Tris-HCl, pH 8.0,100 mm EDTA, 250 mM NaCl, 1% sarkosyl, 1% 2-mercaptoethanol, 10 μg/mlRNase and 12.5 ml phenol] prewarmed to 60° C. was added to the groundtissue. The mixture was shaken for fifteen minutes. An additional 12.5ml of chloroform/isoamyl alcohol (24:1) was added to the mixture andshaken for another 15 minutes. The mixture was centrifuged and theaqueous phase reextracted with 25 ml phenol/chloroform/isoamylalcohol(25:24:1) and chloroform/isoamylalcohol (24:1). The nucleic acids wererecovered by precipitaion with 15 ml isopropanol at room temperature.The precipitate was resuspended in 1 ml of water.

[0173] Genomic DNA was subjected to restriction enzyme digestion asfollows: 10 μg genomic DNA, 40 μl 10×reaction buffer and 100 Urestriction enzyme (XbaI, EcoRI, EcoRV or HinDIII) were reacted for fiveto six hours in a total reaction volume of 400 μl. The mixture was thenphenol-extracted and ethanol-precipitated. The digested DNA wassubjected to agarose gel electrophoresis on a 0.8% agarose gel at 15volts for approximately 15 hours. The gel was submerged in denaturationbuffer [87.66 g NaCl and 20 g NaOH/Liter] for 30 minutes with gentleagitation, rinsed in distilled water and submerged in neutralizationbuffer [87.66 g NaCl and 60.55 g tris-HCl, pH 7.5/Liter] for 30 minuteswith gentle agitation. The DNA was transferred to a Hybond-N⁺ nylonmembrane by capillary blotting.

[0174] Hybridization was performed overnight at 42° C. using 1×10⁶cpm/ml of ³²P-dCTP-labeled full length DHS cDNA or 3′-non-coding regionof the DHS cDNA clone. Prehybridization and hybridization were carriedout in buffer containing 50% formamide, 6×SSC, 5× Denhardt's solution,0.1% SDS and 100 mg/ml denatured salmon sperm DNA. The membrane wasprehybridized for two to four hours; hybridization was carried outovernight.

[0175] After hybridization was complete, membranes were rinsed at roomtemperature in 2×SSC and 0.1% SDS and then washed in 2×SSC and 0.1% SDSfor 15 minutes and 0.2×SSC and 0.1% SDS for 15 minutes. The membrane wasthen exposed to x-ray film at −80° C. overnight. The results are shownin FIG. 5.

EXAMPLE 8

[0176] Isolation of a Senescence-Induced elF-5A Gene from Arabidopsis

[0177] A full-length cDNA clone of the senescence-induced elF-5A geneexpressed in Arabidopsis leaves was obtained by PCR using an Arabidopsissenescing leaf cDNA library as template. Initially, PCR productscorresponding to the 5′- and 3′-ends of the gene were made using adegenerate upstream primer <AAARRYCGMCCYTGCAAGGT> (SEQ ID NO:17) pairedwith vector T7 primer <MTACGACTCACTATAG> (SEQ ID NO:18), and adegenerate downstream primer <TCYTTNCCYTCMKCTMHCC> (SEQ ID NO:19) pairedwith vector T3 primer <ATTAACCCTCACTMAG> (SEQ ID NO: 20). The PCRproducts were subcloned into pBluescript for sequencing. The full-lengthcDNA was then obtained using a 5′-specific primer<CTGTTACCAAAAAATCTGTACC> (SEQ ID NO: 21) paired with a 3′-specificprimer <AGMGAAGTATAAAAACCATC> (SEQ ID NO: 22), and subcloned intopBluescript for sequencing.

EXAMPLE 9

[0178] Isolation of a Senescence-Induced elF-5A Gene from Tomato Fruit

[0179] A full-length cDNA clone of the senescence-induced elF-5A geneexpressed in tomato fruit was obtained by PCR using a tomato fruit cDNAlibrary as template. Initially, PCR products corresponding to the 5′-and 3′-ends of the gene were made using a degenerate upstream primer(SEQ ID NO:17) paired with vector T7 primer (SEQ ID NO:18), and adegenerate downstream primer (SEQ ID NO:19) paired with vector T3 primer(SEQ ID NO: 20). The PCR products were subcloned into pBluescript forsequencing. The full-length cDNA was then obtained using a 5′-specificprimer <AMGAATCCTAGAGAGAGAAAGG> (SEQ ID NO: 23) paired with vector T7primer (SEQ ID NO: 18), and subcloned into pBluescript for sequencing.

EXAMPLE 10

[0180] Isolation of a Senescence-Induced elF-5A Gene from Carnation

[0181] A full-length cDNA clone of the senescence-induced elF-5A geneexpressed in carnation flowers was obtained by PCR using a carnationsenescing flower cDNA library as template. Initially, PCR productscorresponding to the 5′- and 3′-ends of the gene were made using adegenerate upstream primer (SEQ ID NO:17) paired with vector T7 primer(SEQ ID NO:18), and a degenerate downstream primer (SEQ ID NO:19) pairedwith vector T3 primer (SEQ ID NO: 20). The PCR products were subclonedinto pBluescript for sequencing. The full-length cDNA was then obtainedusing a 5′-specific primer <TTTTACATCAATCGAAAA> (SEQ ID NO: 24) pairedwith a 3′-specific primer <ACCAAMCCTGTGTTATMCTCC> (SEQ ID NO: 25), andsubcloned into pBluescript for sequencing.

EXAMPLE 11

[0182] Isolation of a Senescence-Induced DHS Gene from Arabidopsis

[0183] A full-length cDNA clone of the senescence-induced DHS geneexpressed in Arabidopsis leaves was obtained by screening an Arabidopsissenescing leaf cDNA library. The sequence of the probe (SEQ ID NO: 26)that was used for screening is shown in FIG. 38. The probe was obtainedby PCR using the senescence leaf cDNA library as a template and primers(indicated as underlined regions in FIG. 38) designed from theunidentified genomic sequence (AB017060) in GenBank. The PCR product wassubcloned into pBluescript for sequencing.

EXAMPLE 12

[0184] Isolation of a Senescence-Induced DHS Gene from Carnation

[0185] A full-length cDNA clone of the senescence-induced DHS geneexpressed in carnation petals was obtained by screening a carnationsenescing petal cDNA library. The sequence of the probe (SEQ ID NO: 27)that was used for screening is shown in FIG. 39. The probe was obtainedby PCR using the senescence petal cDNA library as a template anddegenerate primers (upstream: 5′ TTG ARG MG ATY CAT MAA RTG CCT 3′) (SEQID NO: 28); downstream: 5′ CCA TCA AAY TCY TGK GCR GTG TT 3′) (SEQ IDNO: 29)). The PCR product was subcloned into pBluescript for sequencing.

EXAMPLE 13

[0186] Transformation of Arabidopsis With Full-Length or 3′ Region ofArabidopsis DHS in Antisense Orientation

[0187] Agrobacteria were transformed with the binary vector, pKYLX71,containing the full-length senescence-induced Arabidopsis DHS cDNAsequence or the 3′ end of the DHS gene (SEQ ID NO:30) (FIG. 36), bothexpressed in the antisense configuration, under the regulation of double35S promoter. Arabidopsis plants were transformed with the transformedAgrobacteria by vacuum infiltration, and transformed seeds fromresultant T₀ plants were selected on ampicillin.

[0188]FIGS. 21 through 24 are photographs of the transformed Arabidopsisplants, showing that expression of the DHS gene or 3′ end thereof inantisense orientation in the transformed plants results in increasedbiomass, e.g., larger leaves and increased plant size. FIG. 25illustrates that the transgenic Arabidopsis plants have increased seedyield.

EXAMPLE 14

[0189] Transformation of Tomato Plants With Full-Length or 3′ Region ofTomato DHS in Antisense Orientation

[0190] Agrobacteria were transformed with the binary vector, pKYLX71,containing the full-length senescence-induced tomato DHS cDNA sequenceor the 3′ end of the DHS gene (SEQ ID NO:31) (FIG. 37), both expressedin the antisense configuration, under the regulation of double 35Spromoter. Tomato leaf explants were formed with these Agrobacteria, andtransformed callus and plantlets were generated and selected by standardtissue culture methods. Transformed plantlets were grown to maturefruit-producing T₁ plants under greenhouse conditions.

[0191]FIGS. 26 through 35 are photographs showing that reducedexpression of the senescence-induced tomato DHS gene in the transformedplants results in increased biomass, e.g., larger leaf size and largerplants as seen in the transformed Arabidopsis plants, as well as delayedsoftening and spoilage of tomato fruit.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 35 <210> SEQ ID NO 1<211> LENGTH: 1609 <212> TYPE: DNA <213> ORGANISM: Lycopersicon sp.<220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (54..1196) <220>FEATURE: <223> OTHER INFORMATION: DHS <400> SEQUENCE: 1 cgcagaaactcgcggcggca gtcttgttcc ctacataatc ttggtctgca ata atg 56 Met 1 gga gaa gctctg aag tac agt atc atg gac tca gta aga tcg gta gtt 104 Gly Glu Ala LeuLys Tyr Ser Ile Met Asp Ser Val Arg Ser Val Val 5 10 15 ttc aaa gaa tccgaa aat cta gaa ggt tct tgc act aaa atc gag ggc 152 Phe Lys Glu Ser GluAsn Leu Glu Gly Ser Cys Thr Lys Ile Glu Gly 20 25 30 tac gac ttc aat aaaggc gtt aac tat gct gag ctg atc aag tcc atg 200 Tyr Asp Phe Asn Lys GlyVal Asn Tyr Ala Glu Leu Ile Lys Ser Met 35 40 45 gtt tcc act ggt ttc caagca tct aat ctt ggt gac gcc att gca att 248 Val Ser Thr Gly Phe Gln AlaSer Asn Leu Gly Asp Ala Ile Ala Ile 50 55 60 65 gtt aat caa atg cta gattgg agg ctt tca cat gag ctg ccc acg gag 296 Val Asn Gln Met Leu Asp TrpArg Leu Ser His Glu Leu Pro Thr Glu 70 75 80 gat tgc agt gaa gaa gaa agagat gtt gca tac aga gag tcg gta acc 344 Asp Cys Ser Glu Glu Glu Arg AspVal Ala Tyr Arg Glu Ser Val Thr 85 90 95 tgc aaa atc ttc ttg ggg ttc acttca aac ctt gtt tct tct ggt gtt 392 Cys Lys Ile Phe Leu Gly Phe Thr SerAsn Leu Val Ser Ser Gly Val 100 105 110 aga gac act gtc cgc tac ctt gttcag cac cgg atg gtt gat gtt gtg 440 Arg Asp Thr Val Arg Tyr Leu Val GlnHis Arg Met Val Asp Val Val 115 120 125 gtt act aca gct ggt ggt att gaagag gat ctc ata aag tgc ctc gca 488 Val Thr Thr Ala Gly Gly Ile Glu GluAsp Leu Ile Lys Cys Leu Ala 130 135 140 145 cca acc tac aag ggg gac ttctct tta cct gga gct tct cta cga tcg 536 Pro Thr Tyr Lys Gly Asp Phe SerLeu Pro Gly Ala Ser Leu Arg Ser 150 155 160 aaa gga ttg aac cgt att ggtaac tta ttg gtt cct aat gac aac tac 584 Lys Gly Leu Asn Arg Ile Gly AsnLeu Leu Val Pro Asn Asp Asn Tyr 165 170 175 tgc aaa ttt gag aat tgg atcatc cca gtt ttt gac caa atg tat gag 632 Cys Lys Phe Glu Asn Trp Ile IlePro Val Phe Asp Gln Met Tyr Glu 180 185 190 gag cag att aat gag aag gttcta tgg aca cca tct aaa gtc att gct 680 Glu Gln Ile Asn Glu Lys Val LeuTrp Thr Pro Ser Lys Val Ile Ala 195 200 205 cgt ctg ggt aaa gaa att aatgat gaa acc tca tac ttg tat tgg gct 728 Arg Leu Gly Lys Glu Ile Asn AspGlu Thr Ser Tyr Leu Tyr Trp Ala 210 215 220 225 tac aag aac cgg att cctgtc ttc tgt cct ggc ttg acg gat gga tca 776 Tyr Lys Asn Arg Ile Pro ValPhe Cys Pro Gly Leu Thr Asp Gly Ser 230 235 240 ctt ggt gac atg cta tacttc cat tct ttc aaa aag ggt gat cca gat 824 Leu Gly Asp Met Leu Tyr PheHis Ser Phe Lys Lys Gly Asp Pro Asp 245 250 255 aat cca gat ctt aat cctggt cta gtc ata gac att gta gga gat att 872 Asn Pro Asp Leu Asn Pro GlyLeu Val Ile Asp Ile Val Gly Asp Ile 260 265 270 agg gcc atg aat ggt gaagct gtc cat gct ggt ttg agg aag aca gga 920 Arg Ala Met Asn Gly Glu AlaVal His Ala Gly Leu Arg Lys Thr Gly 275 280 285 atg att ata ctg ggt ggaggg ctg cct aag cac cat gtt tgc aat gcc 968 Met Ile Ile Leu Gly Gly GlyLeu Pro Lys His His Val Cys Asn Ala 290 295 300 305 aat atg atg cgc aatggt gca gat ttt gcc gtc ttc att aac acc gca 1016 Asn Met Met Arg Asn GlyAla Asp Phe Ala Val Phe Ile Asn Thr Ala 310 315 320 caa gag ttt gat ggtagt gac tct ggt gcc cgt cct gat gaa gct gta 1064 Gln Glu Phe Asp Gly SerAsp Ser Gly Ala Arg Pro Asp Glu Ala Val 325 330 335 tca tgg gga aag atacgt ggt ggt gcc aag act gtg aag gtg cat tgt 1112 Ser Trp Gly Lys Ile ArgGly Gly Ala Lys Thr Val Lys Val His Cys 340 345 350 gat gca acc att gcattt ccc ata tta gta gct gag aca ttt gca gct 1160 Asp Ala Thr Ile Ala PhePro Ile Leu Val Ala Glu Thr Phe Ala Ala 355 360 365 aag agt aag gaa ttctcc cag ata agg tgc caa gtt tgaacattga 1206 Lys Ser Lys Glu Phe Ser GlnIle Arg Cys Gln Val 370 375 380 ggaagctgtc cttccgacca cacatatgaattgctagctt ttgaagccaa cttgctagtg 1266 tgcagcacca tttattctgc aaaactgactagagagcagg gtatattcct ctaccccgag 1326 ttagacgaca tcctgtatgg ttcaaattaattatttttct ccccttcaca ccatgttatt 1386 tagttctctt cctcttcgaa agtgaagagcttagatgttc ataggttttg aattatgttg 1446 gaggttggtg ataactgact agtcctcttaccatatagat aatgtatcct tgtactatga 1506 gattttgggt gtgtttgata ccaaggaaaatgtttatttg gaaaacaatt ggatttttaa 1566 tttatttttt cttgtttaaa aaaaaaaaaaaaaaaaaaaa aaa 1609 <210> SEQ ID NO 2 <211> LENGTH: 381 <212> TYPE: PRT<213> ORGANISM: Lycopersicon sp. <220> FEATURE: <223> OTHER INFORMATION:DHS <400> SEQUENCE: 2 Met Gly Glu Ala Leu Lys Tyr Ser Ile Met Asp SerVal Arg Ser Val 1 5 10 15 Val Phe Lys Glu Ser Glu Asn Leu Glu Gly SerCys Thr Lys Ile Glu 20 25 30 Gly Tyr Asp Phe Asn Lys Gly Val Asn Tyr AlaGlu Leu Ile Lys Ser 35 40 45 Met Val Ser Thr Gly Phe Gln Ala Ser Asn LeuGly Asp Ala Ile Ala 50 55 60 Ile Val Asn Gln Met Leu Asp Trp Arg Leu SerHis Glu Leu Pro Thr 65 70 75 80 Glu Asp Cys Ser Glu Glu Glu Arg Asp ValAla Tyr Arg Glu Ser Val 85 90 95 Thr Cys Lys Ile Phe Leu Gly Phe Thr SerAsn Leu Val Ser Ser Gly 100 105 110 Val Arg Asp Thr Val Arg Tyr Leu ValGln His Arg Met Val Asp Val 115 120 125 Val Val Thr Thr Ala Gly Gly IleGlu Glu Asp Leu Ile Lys Cys Leu 130 135 140 Ala Pro Thr Tyr Lys Gly AspPhe Ser Leu Pro Gly Ala Ser Leu Arg 145 150 155 160 Ser Lys Gly Leu AsnArg Ile Gly Asn Leu Leu Val Pro Asn Asp Asn 165 170 175 Tyr Cys Lys PheGlu Asn Trp Ile Ile Pro Val Phe Asp Gln Met Tyr 180 185 190 Glu Glu GlnIle Asn Glu Lys Val Leu Trp Thr Pro Ser Lys Val Ile 195 200 205 Ala ArgLeu Gly Lys Glu Ile Asn Asp Glu Thr Ser Tyr Leu Tyr Trp 210 215 220 AlaTyr Lys Asn Arg Ile Pro Val Phe Cys Pro Gly Leu Thr Asp Gly 225 230 235240 Ser Leu Gly Asp Met Leu Tyr Phe His Ser Phe Lys Lys Gly Asp Pro 245250 255 Asp Asn Pro Asp Leu Asn Pro Gly Leu Val Ile Asp Ile Val Gly Asp260 265 270 Ile Arg Ala Met Asn Gly Glu Ala Val His Ala Gly Leu Arg LysThr 275 280 285 Gly Met Ile Ile Leu Gly Gly Gly Leu Pro Lys His His ValCys Asn 290 295 300 Ala Asn Met Met Arg Asn Gly Ala Asp Phe Ala Val PheIle Asn Thr 305 310 315 320 Ala Gln Glu Phe Asp Gly Ser Asp Ser Gly AlaArg Pro Asp Glu Ala 325 330 335 Val Ser Trp Gly Lys Ile Arg Gly Gly AlaLys Thr Val Lys Val His 340 345 350 Cys Asp Ala Thr Ile Ala Phe Pro IleLeu Val Ala Glu Thr Phe Ala 355 360 365 Ala Lys Ser Lys Glu Phe Ser GlnIle Arg Cys Gln Val 370 375 380 <210> SEQ ID NO 3 <211> LENGTH: 24 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: primer <400> SEQUENCE:3 agtctagaag gtgctcgtcc tgat 24 <210> SEQ ID NO 4 <211> LENGTH: 34 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: primer <400> SEQUENCE:4 gactgcagtc gacatcgatt tttttttttt tttt 34 <210> SEQ ID NO 5 <211>LENGTH: 2272 <212> TYPE: DNA <213> ORGANISM: Arabidopsis sp. <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (68..265, 348..536,624..842, 979..1065, 1154..1258, 1575..1862) <400> SEQUENCE: 5gaactcccaa aaccctctac tactacactt tcagatccaa ggaaatcaat tttgtcattc 60gagcaac atg gag gat gat cgt gtt ttc tct tcg gtt cac tca aca gtt 109 MetGlu Asp Asp Arg Val Phe Ser Ser Val His Ser Thr Val 1 5 10 ttc aaa gaatcc gaa tca ttg gaa gga aag tgt gat aaa atc gaa gga 157 Phe Lys Glu SerGlu Ser Leu Glu Gly Lys Cys Asp Lys Ile Glu Gly 15 20 25 30 tac gat ttcaat caa gga gta gat tac cca aag ctt atg cga tcc atg 205 Tyr Asp Phe AsnGln Gly Val Asp Tyr Pro Lys Leu Met Arg Ser Met 35 40 45 ctc acc acc ggattt caa gcc tcg aat ctc ggc gaa gct att gat gtc 253 Leu Thr Thr Gly PheGln Ala Ser Asn Leu Gly Glu Ala Ile Asp Val 50 55 60 gtc aat caa atggttcgtttct cgaattcatc aaaaataaaa attccttctt 305 Val Asn Gln Met 65tttgttttcc tttgttttgg gtgaattagt aatgacaaag ag ttt gaa ttt gta 359 PheGlu Phe Val 70 ttg aag cta gat tgg aga ctg gct gat gaa act aca gta gctgaa gac 407 Leu Lys Leu Asp Trp Arg Leu Ala Asp Glu Thr Thr Val Ala GluAsp 75 80 85 tgt agt gaa gag gag aag aat cca tcg ttt aga gag tct gtc aagtgt 455 Cys Ser Glu Glu Glu Lys Asn Pro Ser Phe Arg Glu Ser Val Lys Cys90 95 100 aaa atc ttt cta ggt ttc act tca aat ctt gtt tca tct ggt gttaga 503 Lys Ile Phe Leu Gly Phe Thr Ser Asn Leu Val Ser Ser Gly Val Arg105 110 115 gat act att cgt tat ctt gtt cag cat cat atg gtttgtgatttttgctttat 556 Asp Thr Ile Arg Tyr Leu Val Gln His His Met 120 125caccctgctt ttttatagat gttaaaattt tcgagcttta gttttgattt caatggtttt 616tctgcag gtt gat gtt ata gtc acg aca act ggt ggt gtt gag gaa gat 665 ValAsp Val Ile Val Thr Thr Thr Gly Gly Val Glu Glu Asp 130 135 140 ctc ataaaa tgc ctt gca cct aca ttt aaa ggt gat ttc tct cta cct 713 Leu Ile LysCys Leu Ala Pro Thr Phe Lys Gly Asp Phe Ser Leu Pro 145 150 155 gga gcttat tta agg tca aag gga ttg aac cga att ggg aat ttg ctg 761 Gly Ala TyrLeu Arg Ser Lys Gly Leu Asn Arg Ile Gly Asn Leu Leu 160 165 170 175 gttcct aat gat aac tac tgc aag ttt gag gat tgg atc att ccc atc 809 Val ProAsn Asp Asn Tyr Cys Lys Phe Glu Asp Trp Ile Ile Pro Ile 180 185 190 tttgac gag atg ttg aag gaa cag aaa gaa gag gtattgcttt atctttcctt 862 PheAsp Glu Met Leu Lys Glu Gln Lys Glu Glu 195 200 tttatatgat ttgagatgattctgtttgtg cgtcactagt ggagatagat tttgattcct 922 ctcttgcatc attgacttcgttggtgaatc cttctttctc tggtttttcc ttgtag 978 aat gtg ttg tgg act cct tctaaa ctg tta gca cgg ctg gga aaa gaa 1026 Asn Val Leu Trp Thr Pro Ser LysLeu Leu Ala Arg Leu Gly Lys Glu 205 210 215 atc aac aat gag agt tca tacctt tat tgg gca tac aag gtatccaaaa 1075 Ile Asn Asn Glu Ser Ser Tyr LeuTyr Trp Ala Tyr Lys 220 225 230 ttttaacctt tttagttttt taatcatcctgtgaggaact cggggattta aattttccgc 1135 ttcttgtggt gtttgtag atg aat attcca gta ttc tgc cca ggg tta aca 1186 Met Asn Ile Pro Val Phe Cys Pro GlyLeu Thr 235 240 gat ggc tct ctt ggg gat atg ctg tat ttt cac tct ttt cgtacc tct 1234 Asp Gly Ser Leu Gly Asp Met Leu Tyr Phe His Ser Phe Arg ThrSer 245 250 255 ggc ctc atc atc gat gta gta caa ggtacttctt ttactcaataagtcagtgtg 1288 Gly Leu Ile Ile Asp Val Val Gln 260 265 ataaatattcctgctacatc tagtgcagga atattgtaac tagtagtgca ttgtagcttt 1348 tccaattcagcaacggactt tactgtaagt tgatatctaa aggttcaaac gggagctagg 1408 agaatagcataggggcattc tgatttaggt ttggggcact gggttaagag ttagagaata 1468 ataatcttgttagttgttta tcaaactctt tgatggttag tctcttggta atttgaattt 1528 tatcacagtgtttatggtct ttgaaccagt taatgtttta tgaaca gat atc aga 1583 Asp Ile Arg gctatg aac ggc gaa gct gtc cat gca aat cct aaa aag aca ggg atg 1631 Ala MetAsn Gly Glu Ala Val His Ala Asn Pro Lys Lys Thr Gly Met 270 275 280 285ata atc ctt gga ggg ggc ttg cca aag cac cac ata tgt aat gcc aat 1679 IleIle Leu Gly Gly Gly Leu Pro Lys His His Ile Cys Asn Ala Asn 290 295 300atg atg cgc aat ggt gca gat tac gct gta ttt ata aac acc ggg caa 1727 MetMet Arg Asn Gly Ala Asp Tyr Ala Val Phe Ile Asn Thr Gly Gln 305 310 315gaa ttt gat ggg agc gac tcg ggt gca cgc cct gat gaa gcc gtg tct 1775 GluPhe Asp Gly Ser Asp Ser Gly Ala Arg Pro Asp Glu Ala Val Ser 320 325 330tgg ggt aaa att agg ggt tct gct aaa acc gtt aag gtc tgc ttt tta 1823 TrpGly Lys Ile Arg Gly Ser Ala Lys Thr Val Lys Val Cys Phe Leu 335 340 345att tct tca cat cct aat tta tat ctc act cag tgg ttt tgagtacata 1872 IleSer Ser His Pro Asn Leu Tyr Leu Thr Gln Trp Phe 350 355 360 tttaatattggatcattctt gcaggtatac tgtgatgcta ccatagcctt cccattgttg 1932 gttgcagaaacatttgccac aaagagagac caaacctgtg agtctaagac ttaagaactg 1992 actggtcgttttggccatgg attcttaaag atcgttgctt tttgatttta cactggagtg 2052 accatataacactccacatt gatgtggctg tgacgcgaat tgtcttcttg cgaattgtac 2112 tttagtttctctcaacctaa aatgatttgc agattgtgtt ttcgtttaaa acacaagagt 2172 cttgtagtcaataatccttt gccttataaa attattcagt tccaacaaca cattgtgatt 2232 ctgtgacaagtctcccgttg cctatgttca cttctctgcg 2272 <210> SEQ ID NO 6 <211> LENGTH:362 <212> TYPE: PRT <213> ORGANISM: Arabidopsis sp. <400> SEQUENCE: 6Met Glu Asp Asp Arg Val Phe Ser Ser Val His Ser Thr Val Phe Lys 1 5 1015 Glu Ser Glu Ser Leu Glu Gly Lys Cys Asp Lys Ile Glu Gly Tyr Asp 20 2530 Phe Asn Gln Gly Val Asp Tyr Pro Lys Leu Met Arg Ser Met Leu Thr 35 4045 Thr Gly Phe Gln Ala Ser Asn Leu Gly Glu Ala Ile Asp Val Val Asn 50 5560 Gln Met Phe Glu Phe Val Leu Lys Leu Asp Trp Arg Leu Ala Asp Glu 65 7075 80 Thr Thr Val Ala Glu Asp Cys Ser Glu Glu Glu Lys Asn Pro Ser Phe 8590 95 Arg Glu Ser Val Lys Cys Lys Ile Phe Leu Gly Phe Thr Ser Asn Leu100 105 110 Val Ser Ser Gly Val Arg Asp Thr Ile Arg Tyr Leu Val Gln HisHis 115 120 125 Met Val Asp Val Ile Val Thr Thr Thr Gly Gly Val Glu GluAsp Leu 130 135 140 Ile Lys Cys Leu Ala Pro Thr Phe Lys Gly Asp Phe SerLeu Pro Gly 145 150 155 160 Ala Tyr Leu Arg Ser Lys Gly Leu Asn Arg IleGly Asn Leu Leu Val 165 170 175 Pro Asn Asp Asn Tyr Cys Lys Phe Glu AspTrp Ile Ile Pro Ile Phe 180 185 190 Asp Glu Met Leu Lys Glu Gln Lys GluGlu Asn Val Leu Trp Thr Pro 195 200 205 Ser Lys Leu Leu Ala Arg Leu GlyLys Glu Ile Asn Asn Glu Ser Ser 210 215 220 Tyr Leu Tyr Trp Ala Tyr LysMet Asn Ile Pro Val Phe Cys Pro Gly 225 230 235 240 Leu Thr Asp Gly SerLeu Gly Asp Met Leu Tyr Phe His Ser Phe Arg 245 250 255 Thr Ser Gly LeuIle Ile Asp Val Val Gln Asp Ile Arg Ala Met Asn 260 265 270 Gly Glu AlaVal His Ala Asn Pro Lys Lys Thr Gly Met Ile Ile Leu 275 280 285 Gly GlyGly Leu Pro Lys His His Ile Cys Asn Ala Asn Met Met Arg 290 295 300 AsnGly Ala Asp Tyr Ala Val Phe Ile Asn Thr Gly Gln Glu Phe Asp 305 310 315320 Gly Ser Asp Ser Gly Ala Arg Pro Asp Glu Ala Val Ser Trp Gly Lys 325330 335 Ile Arg Gly Ser Ala Lys Thr Val Lys Val Cys Phe Leu Ile Ser Ser340 345 350 His Pro Asn Leu Tyr Leu Thr Gln Trp Phe 355 360 <210> SEQ IDNO 7 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: primer <400> SEQUENCE: 7 ggtggtgttg aggaagatc 19<210> SEQ ID NO 8 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: primer <400> SEQUENCE: 8 ggtgcacgcc ctgatgaagc20 <210> SEQ ID NO 9 <211> LENGTH: 1660 <212> TYPE: DNA <213> ORGANISM:Dianthus sp. <220> FEATURE: <223> OTHER INFORMATION: DHS <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (256)..(1374) <400> SEQUENCE: 9gtcattacaa tgcataggat cattgcacat gctaccttcc tcattgcact tgagcttgcc 60atacttttgt ttttgacgtt tgataataat actatgaaaa tattatgttt tttcttttgt 120gtgttggtgt ttttgaagtt gtttttgata agcagaaccc agttgtttta cacttttacc 180attgaactac tgcaattcta aaactttgtt tacattttaa ttccatcaaa gattgagttc 240agcataggaa aaagg atg gag gat gct aat cat gat agt gtg gca tct gcg 291 MetGlu Asp Ala Asn His Asp Ser Val Ala Ser Ala 1 5 10 cac tct gca gca ttcaaa aag tcg gag aat tta gag ggg aaa agc gtt 339 His Ser Ala Ala Phe LysLys Ser Glu Asn Leu Glu Gly Lys Ser Val 15 20 25 aag att gag ggt tat gatttt aat caa ggt gta aac tat tcc aaa ctc 387 Lys Ile Glu Gly Tyr Asp PheAsn Gln Gly Val Asn Tyr Ser Lys Leu 30 35 40 ttg caa tct ttc gct tct aatggg ttt caa gcc tcg aat ctt gga gat 435 Leu Gln Ser Phe Ala Ser Asn GlyPhe Gln Ala Ser Asn Leu Gly Asp 45 50 55 60 gcc att gaa gta gtt aat catatg cta gat tgg agt ctg gca gat gag 483 Ala Ile Glu Val Val Asn His MetLeu Asp Trp Ser Leu Ala Asp Glu 65 70 75 gca cct gtg gac gat tgt agc gaggaa gag agg gat cct aaa ttc aga 531 Ala Pro Val Asp Asp Cys Ser Glu GluGlu Arg Asp Pro Lys Phe Arg 80 85 90 gaa tct gtg aag tgc aaa gtg ttc ttgggc ttt act tca aat ctt att 579 Glu Ser Val Lys Cys Lys Val Phe Leu GlyPhe Thr Ser Asn Leu Ile 95 100 105 tcc tct ggt gtt cgt gac aca att cggtat ctc gtg caa cat cat atg 627 Ser Ser Gly Val Arg Asp Thr Ile Arg TyrLeu Val Gln His His Met 110 115 120 gtt gac gtg ata gta acg aca acc ggaggt ata gaa gaa gat cta ata 675 Val Asp Val Ile Val Thr Thr Thr Gly GlyIle Glu Glu Asp Leu Ile 125 130 135 140 aaa gga aga tcc atc aag tgc cttgca ccc act ttc aaa ggc gat ttt 723 Lys Gly Arg Ser Ile Lys Cys Leu AlaPro Thr Phe Lys Gly Asp Phe 145 150 155 gcc tta cca gga gct caa tta cgctcc aaa ggg ttg aat cga att ggt 771 Ala Leu Pro Gly Ala Gln Leu Arg SerLys Gly Leu Asn Arg Ile Gly 160 165 170 aat ctg ttg gtt ccg aat gat aactac tgt aaa ttt gag gat tgg atc 819 Asn Leu Leu Val Pro Asn Asp Asn TyrCys Lys Phe Glu Asp Trp Ile 175 180 185 att cca att tta gat aag atg ttggaa gag caa att tca gag aaa atc 867 Ile Pro Ile Leu Asp Lys Met Leu GluGlu Gln Ile Ser Glu Lys Ile 190 195 200 tta tgg aca cca tcg aag ttg attggt cga tta gga aga gaa ata aac 915 Leu Trp Thr Pro Ser Lys Leu Ile GlyArg Leu Gly Arg Glu Ile Asn 205 210 215 220 gat gag agt tca tac ctt tactgg gcc ttc aag aac aat att cca gta 963 Asp Glu Ser Ser Tyr Leu Tyr TrpAla Phe Lys Asn Asn Ile Pro Val 225 230 235 ttt tgc cca ggt tta aca gacggc tca ctc gga gac atg cta tat ttt 1011 Phe Cys Pro Gly Leu Thr Asp GlySer Leu Gly Asp Met Leu Tyr Phe 240 245 250 cat tct ttt cgc aat ccg ggttta atc gtc gat gtt gtg caa gat ata 1059 His Ser Phe Arg Asn Pro Gly LeuIle Val Asp Val Val Gln Asp Ile 255 260 265 aga gca gta aat ggc gag gctgtg cac gca gcg cct agg aaa aca ggc 1107 Arg Ala Val Asn Gly Glu Ala ValHis Ala Ala Pro Arg Lys Thr Gly 270 275 280 atg att ata ctc ggt gga gggttg cct aag cac cac atc tgc aac gca 1155 Met Ile Ile Leu Gly Gly Gly LeuPro Lys His His Ile Cys Asn Ala 285 290 295 300 aac atg atg aga aat ggcgcc gat tat gct gtt ttc atc aac act gcc 1203 Asn Met Met Arg Asn Gly AlaAsp Tyr Ala Val Phe Ile Asn Thr Ala 305 310 315 gaa gag ttt gac ggc agtgat tct ggt gct cgc ccc gat gag gct att 1251 Glu Glu Phe Asp Gly Ser AspSer Gly Ala Arg Pro Asp Glu Ala Ile 320 325 330 tca tgg ggc aaa att agcgga tct gct aag act gtg aag gtg cat tgt 1299 Ser Trp Gly Lys Ile Ser GlySer Ala Lys Thr Val Lys Val His Cys 335 340 345 gat gcc acg ata gct ttccct cta cta gtc gct gag aca ttt gca gca 1347 Asp Ala Thr Ile Ala Phe ProLeu Leu Val Ala Glu Thr Phe Ala Ala 350 355 360 aaa aga gaa aaa gag aggaag agc tgt taaaactttt ttgattgttg 1394 Lys Arg Glu Lys Glu Arg Lys SerCys 365 370 aaaaatctgt gttatacaag tctcgaaatg cattttagta attgacttgatcttatcatt 1454 tcaatgtgtt atctttgaaa atgttggtaa tgaaacatct cacctcttctatacaacatt 1514 gttgatccat tgtactccgt atcttgtaat tttggaaaaa aaaaaccgtctattgttacg 1574 agagagtaca tttttgaggt aaaaatatag gatttttgtg cgatgcaaatgctggttatt 1634 cccttgaaaa aaaaaaaaaa aaaaaa 1660 <210> SEQ ID NO 10<211> LENGTH: 373 <212> TYPE: PRT <213> ORGANISM: Dianthus sp. <220>FEATURE: <223> OTHER INFORMATION: DHS <400> SEQUENCE: 10 Met Glu Asp AlaAsn His Asp Ser Val Ala Ser Ala His Ser Ala Ala 1 5 10 15 Phe Lys LysSer Glu Asn Leu Glu Gly Lys Ser Val Lys Ile Glu Gly 20 25 30 Tyr Asp PheAsn Gln Gly Val Asn Tyr Ser Lys Leu Leu Gln Ser Phe 35 40 45 Ala Ser AsnGly Phe Gln Ala Ser Asn Leu Gly Asp Ala Ile Glu Val 50 55 60 Val Asn HisMet Leu Asp Trp Ser Leu Ala Asp Glu Ala Pro Val Asp 65 70 75 80 Asp CysSer Glu Glu Glu Arg Asp Pro Lys Phe Arg Glu Ser Val Lys 85 90 95 Cys LysVal Phe Leu Gly Phe Thr Ser Asn Leu Ile Ser Ser Gly Val 100 105 110 ArgAsp Thr Ile Arg Tyr Leu Val Gln His His Met Val Asp Val Ile 115 120 125Val Thr Thr Thr Gly Gly Ile Glu Glu Asp Leu Ile Lys Gly Arg Ser 130 135140 Ile Lys Cys Leu Ala Pro Thr Phe Lys Gly Asp Phe Ala Leu Pro Gly 145150 155 160 Ala Gln Leu Arg Ser Lys Gly Leu Asn Arg Ile Gly Asn Leu LeuVal 165 170 175 Pro Asn Asp Asn Tyr Cys Lys Phe Glu Asp Trp Ile Ile ProIle Leu 180 185 190 Asp Lys Met Leu Glu Glu Gln Ile Ser Glu Lys Ile LeuTrp Thr Pro 195 200 205 Ser Lys Leu Ile Gly Arg Leu Gly Arg Glu Ile AsnAsp Glu Ser Ser 210 215 220 Tyr Leu Tyr Trp Ala Phe Lys Asn Asn Ile ProVal Phe Cys Pro Gly 225 230 235 240 Leu Thr Asp Gly Ser Leu Gly Asp MetLeu Tyr Phe His Ser Phe Arg 245 250 255 Asn Pro Gly Leu Ile Val Asp ValVal Gln Asp Ile Arg Ala Val Asn 260 265 270 Gly Glu Ala Val His Ala AlaPro Arg Lys Thr Gly Met Ile Ile Leu 275 280 285 Gly Gly Gly Leu Pro LysHis His Ile Cys Asn Ala Asn Met Met Arg 290 295 300 Asn Gly Ala Asp TyrAla Val Phe Ile Asn Thr Ala Glu Glu Phe Asp 305 310 315 320 Gly Ser AspSer Gly Ala Arg Pro Asp Glu Ala Ile Ser Trp Gly Lys 325 330 335 Ile SerGly Ser Ala Lys Thr Val Lys Val His Cys Asp Ala Thr Ile 340 345 350 AlaPhe Pro Leu Leu Val Ala Glu Thr Phe Ala Ala Lys Arg Glu Lys 355 360 365Glu Arg Lys Ser Cys 370 <210> SEQ ID NO 11 <211> LENGTH: 780 <212> TYPE:DNA <213> ORGANISM: Lycopersicon sp. <220> FEATURE: <223> OTHERINFORMATION: eif-5A <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION:(43)..(522) <400> SEQUENCE: 11 aaagaatcct agagagagaa agggaatcctagagagagaa gc atg tcg gac gaa 54 Met Ser Asp Glu 1 gaa cac cat ttt gagtca aag gca gat gct ggt gcc tca aaa act ttc 102 Glu His His Phe Glu SerLys Ala Asp Ala Gly Ala Ser Lys Thr Phe 5 10 15 20 cca cag caa gct ggaacc atc cgt aag aat ggt tac atc gtt atc aaa 150 Pro Gln Gln Ala Gly ThrIle Arg Lys Asn Gly Tyr Ile Val Ile Lys 25 30 35 ggc cgt ccc tgc aag gttgtt gag gtc tcc act tca aaa act gga aaa 198 Gly Arg Pro Cys Lys Val ValGlu Val Ser Thr Ser Lys Thr Gly Lys 40 45 50 cac gga cat gct aaa tgt cacttt gtg gca att gac att ttc aat gga 246 His Gly His Ala Lys Cys His PheVal Ala Ile Asp Ile Phe Asn Gly 55 60 65 aag aaa ctg gaa gat atc gtt ccgtcc tcc cac aat tgt gat gtg cca 294 Lys Lys Leu Glu Asp Ile Val Pro SerSer His Asn Cys Asp Val Pro 70 75 80 cat gtt aac cgt acc gac tat cag ctgatt gat atc tct gaa gat ggt 342 His Val Asn Arg Thr Asp Tyr Gln Leu IleAsp Ile Ser Glu Asp Gly 85 90 95 100 ttt gtc tca ctt ctt act gaa agt ggaaac acc aag gat gac ctc agg 390 Phe Val Ser Leu Leu Thr Glu Ser Gly AsnThr Lys Asp Asp Leu Arg 105 110 115 ctt ccc acc gat gaa aat ctg ctg aagcag gtt aaa gat ggg ttc cag 438 Leu Pro Thr Asp Glu Asn Leu Leu Lys GlnVal Lys Asp Gly Phe Gln 120 125 130 gaa gga aag gat ctt gtg gtg tct gttatg tct gcg atg ggc gaa gag 486 Glu Gly Lys Asp Leu Val Val Ser Val MetSer Ala Met Gly Glu Glu 135 140 145 cag att aac gcc gtt aag gat gtt ggtacc aag aat tagttatgtc 532 Gln Ile Asn Ala Val Lys Asp Val Gly Thr LysAsn 150 155 160 atggcagcat aatcactgcc aaagctttaa gacattatca tatcctaatgtggtactttg 592 atatcactag attataaact gtgttatttg cactgttcaa aacaaaagaaagaaaactgc 652 tgttatggct agagaaagta ttggctttga gcttttgaca gcacagttgaactatgtgaa 712 aattctactt tttttttttt gggtaaaata ctgctcgttt aatgttttgcaaaaaaaaaa 772 aaaaaaaa 780 <210> SEQ ID NO 12 <211> LENGTH: 160 <212>TYPE: PRT <213> ORGANISM: Lycopersicon sp. <220> FEATURE: <223> OTHERINFORMATION: eif-5A <400> SEQUENCE: 12 Met Ser Asp Glu Glu His His PheGlu Ser Lys Ala Asp Ala Gly Ala 1 5 10 15 Ser Lys Thr Phe Pro Gln GlnAla Gly Thr Ile Arg Lys Asn Gly Tyr 20 25 30 Ile Val Ile Lys Gly Arg ProCys Lys Val Val Glu Val Ser Thr Ser 35 40 45 Lys Thr Gly Lys His Gly HisAla Lys Cys His Phe Val Ala Ile Asp 50 55 60 Ile Phe Asn Gly Lys Lys LeuGlu Asp Ile Val Pro Ser Ser His Asn 65 70 75 80 Cys Asp Val Pro His ValAsn Arg Thr Asp Tyr Gln Leu Ile Asp Ile 85 90 95 Ser Glu Asp Gly Phe ValSer Leu Leu Thr Glu Ser Gly Asn Thr Lys 100 105 110 Asp Asp Leu Arg LeuPro Thr Asp Glu Asn Leu Leu Lys Gln Val Lys 115 120 125 Asp Gly Phe GlnGlu Gly Lys Asp Leu Val Val Ser Val Met Ser Ala 130 135 140 Met Gly GluGlu Gln Ile Asn Ala Val Lys Asp Val Gly Thr Lys Asn 145 150 155 160<210> SEQ ID NO 13 <211> LENGTH: 812 <212> TYPE: DNA <213> ORGANISM:Dianthus sp. <220> FEATURE: <223> OTHER INFORMATION: eif-5A <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (67)..(546) <400> SEQUENCE:13 ctcttttaca tcaatcgaaa aaaaattagg gttcttattt tagagtgaga ggcgaaaaat 60cgaacg atg tcg gac gac gat cac cat ttc gag tca tcg gcc gac gcc 108 MetSer Asp Asp Asp His His Phe Glu Ser Ser Ala Asp Ala 1 5 10 gga gca tccaag act tac cct caa caa gct ggt aca atc cgc aag agc 156 Gly Ala Ser LysThr Tyr Pro Gln Gln Ala Gly Thr Ile Arg Lys Ser 15 20 25 30 ggt cac atcgtc atc aaa aat cgc cct tgc aag gtg gtt gag gtt tct 204 Gly His Ile ValIle Lys Asn Arg Pro Cys Lys Val Val Glu Val Ser 35 40 45 acc tcc aag actggc aag cac ggt cat gcc aaa tgt cac ttt gtt gcc 252 Thr Ser Lys Thr GlyLys His Gly His Ala Lys Cys His Phe Val Ala 50 55 60 att gac att ttc aacggc aag aag ctg gaa gat att gtc ccc tca tcc 300 Ile Asp Ile Phe Asn GlyLys Lys Leu Glu Asp Ile Val Pro Ser Ser 65 70 75 cac aat tgt gat gtt ccacat gtc aac cgt gtc gac tac cag ctg ctt 348 His Asn Cys Asp Val Pro HisVal Asn Arg Val Asp Tyr Gln Leu Leu 80 85 90 gat atc act gaa gat ggc tttgtt agt ctg ctg act gac agt ggt gac 396 Asp Ile Thr Glu Asp Gly Phe ValSer Leu Leu Thr Asp Ser Gly Asp 95 100 105 110 acc aag gat gat ctg aagctt cct gct gat gag gcc ctt gtg aag cag 444 Thr Lys Asp Asp Leu Lys LeuPro Ala Asp Glu Ala Leu Val Lys Gln 115 120 125 atg aag gag gga ttt gaggcg ggg aaa gac ttg att ctg tca gtc atg 492 Met Lys Glu Gly Phe Glu AlaGly Lys Asp Leu Ile Leu Ser Val Met 130 135 140 tgt gca atg gga gaa gagcag atc tgc gcc gtc aag gac gtt agt ggt 540 Cys Ala Met Gly Glu Glu GlnIle Cys Ala Val Lys Asp Val Ser Gly 145 150 155 ggc aag tagaagcttttgatgaatcc aatactacgc ggtgcagttg aagcaatagt 596 Gly Lys 160 aatctcgagaacattctgaa ccttatatgt tgaattgatg gtgcttagtt tgttttggaa 656 atctctttgcaattaagttg taccaaatca atggatgtaa tgtcttgaat ttgttttatt 716 tttgttttgatgtttgctgt gattgcatta tgcattgtta tgagttatga cctgttataa 776 cacaaggttttggtaaaaaa aaaaaaaaaa aaaaaa 812 <210> SEQ ID NO 14 <211> LENGTH: 160<212> TYPE: PRT <213> ORGANISM: Dianthus sp. <220> FEATURE: <223> OTHERINFORMATION: eif-5A <400> SEQUENCE: 14 Met Ser Asp Asp Asp His His PheGlu Ser Ser Ala Asp Ala Gly Ala 1 5 10 15 Ser Lys Thr Tyr Pro Gln GlnAla Gly Thr Ile Arg Lys Ser Gly His 20 25 30 Ile Val Ile Lys Asn Arg ProCys Lys Val Val Glu Val Ser Thr Ser 35 40 45 Lys Thr Gly Lys His Gly HisAla Lys Cys His Phe Val Ala Ile Asp 50 55 60 Ile Phe Asn Gly Lys Lys LeuGlu Asp Ile Val Pro Ser Ser His Asn 65 70 75 80 Cys Asp Val Pro His ValAsn Arg Val Asp Tyr Gln Leu Leu Asp Ile 85 90 95 Thr Glu Asp Gly Phe ValSer Leu Leu Thr Asp Ser Gly Asp Thr Lys 100 105 110 Asp Asp Leu Lys LeuPro Ala Asp Glu Ala Leu Val Lys Gln Met Lys 115 120 125 Glu Gly Phe GluAla Gly Lys Asp Leu Ile Leu Ser Val Met Cys Ala 130 135 140 Met Gly GluGlu Gln Ile Cys Ala Val Lys Asp Val Ser Gly Gly Lys 145 150 155 160<210> SEQ ID NO 15 <211> LENGTH: 702 <212> TYPE: DNA <213> ORGANISM:Arabidopsis sp. <220> FEATURE: <223> OTHER INFORMATION: eif-5A <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (56)..(529) <400> SEQUENCE:15 ctgttaccaa aaaatctgta ccgcaaaatc ctcgtcgaag ctcgctgctg caacc atg 58Met 1 tcc gac gag gag cat cac ttt gag tcc agt gac gcc gga gcg tcc aaa106 Ser Asp Glu Glu His His Phe Glu Ser Ser Asp Ala Gly Ala Ser Lys 5 1015 acc tac cct caa caa gct gga acc atc cgt aag aat ggt tac atc gtc 154Thr Tyr Pro Gln Gln Ala Gly Thr Ile Arg Lys Asn Gly Tyr Ile Val 20 25 30atc aaa aat cgt ccc tgc aag gtt gtt gag gtt tca acc tcg aag act 202 IleLys Asn Arg Pro Cys Lys Val Val Glu Val Ser Thr Ser Lys Thr 35 40 45 ggcaag cat ggt cat gct aaa tgt cat ttt gta gct att gat atc ttc 250 Gly LysHis Gly His Ala Lys Cys His Phe Val Ala Ile Asp Ile Phe 50 55 60 65 accagc aag aaa ctc gaa gat att gtt cct tct tcc cac aat tgt gat 298 Thr SerLys Lys Leu Glu Asp Ile Val Pro Ser Ser His Asn Cys Asp 70 75 80 gtt cctcat gtc aac cgt act gat tat cag ctg att gac att tct gaa 346 Val Pro HisVal Asn Arg Thr Asp Tyr Gln Leu Ile Asp Ile Ser Glu 85 90 95 gat gga tatgtc agt ttg ttg act gat aac ggt agt acc aag gat gac 394 Asp Gly Tyr ValSer Leu Leu Thr Asp Asn Gly Ser Thr Lys Asp Asp 100 105 110 ctt aag ctccct aat gat gac act ctg ctc caa cag atc aag agt ggg 442 Leu Lys Leu ProAsn Asp Asp Thr Leu Leu Gln Gln Ile Lys Ser Gly 115 120 125 ttt gat gatgga aaa gat cta gtg gtg agt gta atg tca gct atg gga 490 Phe Asp Asp GlyLys Asp Leu Val Val Ser Val Met Ser Ala Met Gly 130 135 140 145 gag gaacag atc aat gct ctt aag gac atc ggt ccc aag tgagactaac 539 Glu Glu GlnIle Asn Ala Leu Lys Asp Ile Gly Pro Lys 150 155 aaagcctccc ctttgttatgagattcttct tcttctgtag gcttccatta ctcgtcggag 599 attatcttgt ttttgggttactcctatttt ggatatttaa acttttgtta ataatgccat 659 cttcttcaac cttttccttctagatggttt ttatacttct tct 702 <210> SEQ ID NO 16 <211> LENGTH: 158 <212>TYPE: PRT <213> ORGANISM: Arabidopsis sp. <220> FEATURE: <223> OTHERINFORMATION: eif-5A <400> SEQUENCE: 16 Met Ser Asp Glu Glu His His PheGlu Ser Ser Asp Ala Gly Ala Ser 1 5 10 15 Lys Thr Tyr Pro Gln Gln AlaGly Thr Ile Arg Lys Asn Gly Tyr Ile 20 25 30 Val Ile Lys Asn Arg Pro CysLys Val Val Glu Val Ser Thr Ser Lys 35 40 45 Thr Gly Lys His Gly His AlaLys Cys His Phe Val Ala Ile Asp Ile 50 55 60 Phe Thr Ser Lys Lys Leu GluAsp Ile Val Pro Ser Ser His Asn Cys 65 70 75 80 Asp Val Pro His Val AsnArg Thr Asp Tyr Gln Leu Ile Asp Ile Ser 85 90 95 Glu Asp Gly Tyr Val SerLeu Leu Thr Asp Asn Gly Ser Thr Lys Asp 100 105 110 Asp Leu Lys Leu ProAsn Asp Asp Thr Leu Leu Gln Gln Ile Lys Ser 115 120 125 Gly Phe Asp AspGly Lys Asp Leu Val Val Ser Val Met Ser Ala Met 130 135 140 Gly Glu GluGln Ile Asn Ala Leu Lys Asp Ile Gly Pro Lys 145 150 155 <210> SEQ ID NO17 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: primer <400> SEQUENCE: 17 aaarrycgmc cytgcaaggt 20 <210> SEQID NO 18 <211> LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: primer <400> SEQUENCE: 18 aatacgactc actatag 17<210> SEQ ID NO 19 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: primer <220> FEATURE: <223> OTHER INFORMATION:“n” bases represent a, t, c, g, other or unknown <400> SEQUENCE: 19tcyttnccyt cmkctaahcc 20 <210> SEQ ID NO 20 <211> LENGTH: 17 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: primer <400> SEQUENCE:20 attaaccctc actaaag 17 <210> SEQ ID NO 21 <211> LENGTH: 22 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: primer <400> SEQUENCE:21 ctgttaccaa aaaatctgta cc 22 <210> SEQ ID NO 22 <211> LENGTH: 21 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: primer <400> SEQUENCE:22 agaagaagta taaaaaccat c 21 <210> SEQ ID NO 23 <211> LENGTH: 23 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: primer <400> SEQUENCE:23 aaagaatcct agagagagaa agg 23 <210> SEQ ID NO 24 <211> LENGTH: 18<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence: primer <400>SEQUENCE: 24 ttttacatca atcgaaaa 18 <210> SEQ ID NO 25 <211> LENGTH: 23<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence: primer <400>SEQUENCE: 25 accaaaacct gtgttataac tcc 23 <210> SEQ ID NO 26 <211>LENGTH: 581 <212> TYPE: DNA <213> ORGANISM: Arabidopsis sp. <220>FEATURE: <223> OTHER INFORMATION: DHS <220> FEATURE: <221> NAME/KEY: CDS<222> LOCATION: (1)..(579) <400> SEQUENCE: 26 ggt ggt gtt gag gaa gatctc ata aaa tgc ctt gca cct aca ttt aaa 48 Gly Gly Val Glu Glu Asp LeuIle Lys Cys Leu Ala Pro Thr Phe Lys 1 5 10 15 ggt gat ttc tct cta cctgga gct tat tta agg tca aag gga ttg aac 96 Gly Asp Phe Ser Leu Pro GlyAla Tyr Leu Arg Ser Lys Gly Leu Asn 20 25 30 cga att ggg aat ttg ctg gttcct aat gat aac tac tgc aag ttt gag 144 Arg Ile Gly Asn Leu Leu Val ProAsn Asp Asn Tyr Cys Lys Phe Glu 35 40 45 gat tgg atc att ccc atc ttt gacgag atg ttg aag gaa cag aaa gaa 192 Asp Trp Ile Ile Pro Ile Phe Asp GluMet Leu Lys Glu Gln Lys Glu 50 55 60 gag aat gtg ttg tgg act cct tct aaactg tta gca cgg ctg gga aaa 240 Glu Asn Val Leu Trp Thr Pro Ser Lys LeuLeu Ala Arg Leu Gly Lys 65 70 75 80 gaa atc aac aat gag agt tca tac ctttat tgg gca tac aag atg aat 288 Glu Ile Asn Asn Glu Ser Ser Tyr Leu TyrTrp Ala Tyr Lys Met Asn 85 90 95 att cca gta ttc tgc cca ggg tta aca gatggc tct ctt agg gat atg 336 Ile Pro Val Phe Cys Pro Gly Leu Thr Asp GlySer Leu Arg Asp Met 100 105 110 ctg tat ttt cac tct ttt cgt acc tct ggcctc atc atc gat gta gta 384 Leu Tyr Phe His Ser Phe Arg Thr Ser Gly LeuIle Ile Asp Val Val 115 120 125 caa gat atc aga gct atg aac ggc gaa gctgtc cat gca aat cct aaa 432 Gln Asp Ile Arg Ala Met Asn Gly Glu Ala ValHis Ala Asn Pro Lys 130 135 140 aag aca ggg atg ata atc ctt gga ggg ggcttg cca aag cac cac ata 480 Lys Thr Gly Met Ile Ile Leu Gly Gly Gly LeuPro Lys His His Ile 145 150 155 160 tgt aat gcc aat atg atg cgc aat ggtgca gat tac gct gta ttt ata 528 Cys Asn Ala Asn Met Met Arg Asn Gly AlaAsp Tyr Ala Val Phe Ile 165 170 175 aac acc ggg caa gaa ttt gat ggg agcgac tcg ggt gca cgc cct gat 576 Asn Thr Gly Gln Glu Phe Asp Gly Ser AspSer Gly Ala Arg Pro Asp 180 185 190 gaa gc 581 Glu <210> SEQ ID NO 27<211> LENGTH: 522 <212> TYPE: DNA <213> ORGANISM: Dianthus sp. <220>FEATURE: <223> OTHER INFORMATION: DHS <220> FEATURE: <221> NAME/KEY: CDS<222> LOCATION: (3)..(521) <400> SEQUENCE: 27 ga aga tcc atc aag tgc cttgca ccc act ttc aaa ggc gat ttt gcc 47 Arg Ser Ile Lys Cys Leu Ala ProThr Phe Lys Gly Asp Phe Ala 1 5 10 15 tta cca gga gct caa tta cgc tccaaa ggg ttg aat cga att ggt aat 95 Leu Pro Gly Ala Gln Leu Arg Ser LysGly Leu Asn Arg Ile Gly Asn 20 25 30 ctg ttg gtt ccg aat gat aac tac tgtaaa ttt gag gat tgg atc att 143 Leu Leu Val Pro Asn Asp Asn Tyr Cys LysPhe Glu Asp Trp Ile Ile 35 40 45 cca att tta gat aag atg ttg gaa gag caaatt tca gag aaa atc tta 191 Pro Ile Leu Asp Lys Met Leu Glu Glu Gln IleSer Glu Lys Ile Leu 50 55 60 tgg aca cca tcg aag ttg att ggt cga tta ggaaga gaa ata aac gat 239 Trp Thr Pro Ser Lys Leu Ile Gly Arg Leu Gly ArgGlu Ile Asn Asp 65 70 75 gag agt tca tac ctt tac tgg gcc ttc aag aac aatatt cca gta ttt 287 Glu Ser Ser Tyr Leu Tyr Trp Ala Phe Lys Asn Asn IlePro Val Phe 80 85 90 95 tgc cca ggt tta aca gac ggc tca ctc gga gac atgcta tat ttt cat 335 Cys Pro Gly Leu Thr Asp Gly Ser Leu Gly Asp Met LeuTyr Phe His 100 105 110 tct ttt cgc aat ccg ggt tta atc atc gat gtt gtgcaa gat ata aga 383 Ser Phe Arg Asn Pro Gly Leu Ile Ile Asp Val Val GlnAsp Ile Arg 115 120 125 gca gta aat ggc gag gct gtg cac gca gcg cct aggaaa aca ggc atg 431 Ala Val Asn Gly Glu Ala Val His Ala Ala Pro Arg LysThr Gly Met 130 135 140 att ata ctc ggt gga ggg ttg cct aag cac cac atctgc aac gca aac 479 Ile Ile Leu Gly Gly Gly Leu Pro Lys His His Ile CysAsn Ala Asn 145 150 155 atg atg aga aat ggc gcc gat tat gct gtt ttc atcaac acc g 522 Met Met Arg Asn Gly Ala Asp Tyr Ala Val Phe Ile Asn Thr160 165 170 <210> SEQ ID NO 28 <211> LENGTH: 24 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: primer <400> SEQUENCE: 28 ttgargaagatycatmaart gcct 24 <210> SEQ ID NO 29 <211> LENGTH: 23 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: primer <400> SEQUENCE:29 ccatcaaayt cytgkgcrgt gtt 23 <210> SEQ ID NO 30 <211> LENGTH: 484<212> TYPE: DNA <213> ORGANISM: Arabidopsis sp. <220> FEATURE: <223>OTHER INFORMATION: DHS <220> FEATURE: <221> NAME/KEY: CDS <222>LOCATION: (2)..(112) <400> SEQUENCE: 30 t gca cgc cct gat gaa gct gtgtct tgg ggt aaa att agg ggt tct gct 49 Ala Arg Pro Asp Glu Ala Val SerTrp Gly Lys Ile Arg Gly Ser Ala 1 5 10 15 aaa acc gtt aag gtc tgc ttttta att tct tca cat cct aat tta tat 97 Lys Thr Val Lys Val Cys Phe LeuIle Ser Ser His Pro Asn Leu Tyr 20 25 30 ctc act cag tgg ttt tgagtacatatttaatattg gatcattctt gcaggtatac 152 Leu Thr Gln Trp Phe 35 tgtgatgctaccatagcctt cccattgttg gttgcagaaa catttgccac aaagagagac 212 caaacctgtgagtctaagac ttaagaactg actggtcgtt ttggccatgg attcttaaag 272 atcgttgctttttgatttta cactggagtg accatataac actccacatt gatgtggctg 332 tgacgcgaattgtcttcttg cgaattgtac tttagtttct ctcaacctaa aatgatttgc 392 agattgtgttttcgtttaaa acacaagagt cttgtagtca ataatccttt gccttataaa 452 attattcagttccaacaaaa aaaaaaaaaa aa 484 <210> SEQ ID NO 31 <211> LENGTH: 559 <212>TYPE: DNA <213> ORGANISM: Lycopersicon sp. <220> FEATURE: <223> OTHERINFORMATION: DHS <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION:(1)..(156) <220> FEATURE: <223> OTHER INFORMATION: “n” bases representa, t, c, g, other or unknown <400> SEQUENCE: 31 ggt gct cgt cct gat gaagct gta tca tgg gga aag ata cgt ggt ggt 48 Gly Ala Arg Pro Asp Glu AlaVal Ser Trp Gly Lys Ile Arg Gly Gly 1 5 10 15 gcc aag act gtg aag gtgcat tgt gat gca acc att gca ttt ccc ata 96 Ala Lys Thr Val Lys Val HisCys Asp Ala Thr Ile Ala Phe Pro Ile 20 25 30 tta gta gct gag aca ttt gcagct aag agt aag gaa ttc tcc cag ata 144 Leu Val Ala Glu Thr Phe Ala AlaLys Ser Lys Glu Phe Ser Gln Ile 35 40 45 agg tgc caa gtt tgaacattgaggaagctgtc cttccgacca cacatatgaa 196 Arg Cys Gln Val 50 ttgctagcttttgaagccaa cttgctagtg tgcagcacca tttattctgc aaaactgact 256 agagagcagggtatattcct ctaccccgag ttagacgaca tcctgtatgg ttcaaattaa 316 ttatttttctccccttcaca ccatgttatt tagttctctt cctcttcgaa agtgaagagc 376 ttagatgttcataggttttg aattatgttg gaggttggtg ataactgact agtcctctta 436 ccatatagataatgtatcct tgtactatga gattttgggt gtgtttgata ccaaggaaaa 496 atgtttatttggaaaacaat tggattttta atttaaaaaa aattgnttaa aaaaaaaaaa 556 aaa 559 <210>SEQ ID NO 32 <211> LENGTH: 193 <212> TYPE: PRT <213> ORGANISM:Arabidopsis sp. <220> FEATURE: <223> OTHER INFORMATION: DHS <400>SEQUENCE: 32 Gly Gly Val Glu Glu Asp Leu Ile Lys Cys Leu Ala Pro Thr PheLys 1 5 10 15 Gly Asp Phe Ser Leu Pro Gly Ala Tyr Leu Arg Ser Lys GlyLeu Asn 20 25 30 Arg Ile Gly Asn Leu Leu Val Pro Asn Asp Asn Tyr Cys LysPhe Glu 35 40 45 Asp Trp Ile Ile Pro Ile Phe Asp Glu Met Leu Lys Glu GlnLys Glu 50 55 60 Glu Asn Val Leu Trp Thr Pro Ser Lys Leu Leu Ala Arg LeuGly Lys 65 70 75 80 Glu Ile Asn Asn Glu Ser Ser Tyr Leu Tyr Trp Ala TyrLys Met Asn 85 90 95 Ile Pro Val Phe Cys Pro Gly Leu Thr Asp Gly Ser LeuArg Asp Met 100 105 110 Leu Tyr Phe His Ser Phe Arg Thr Ser Gly Leu IleIle Asp Val Val 115 120 125 Gln Asp Ile Arg Ala Met Asn Gly Glu Ala ValHis Ala Asn Pro Lys 130 135 140 Lys Thr Gly Met Ile Ile Leu Gly Gly GlyLeu Pro Lys His His Ile 145 150 155 160 Cys Asn Ala Asn Met Met Arg AsnGly Ala Asp Tyr Ala Val Phe Ile 165 170 175 Asn Thr Gly Gln Glu Phe AspGly Ser Asp Ser Gly Ala Arg Pro Asp 180 185 190 Glu <210> SEQ ID NO 33<211> LENGTH: 173 <212> TYPE: PRT <213> ORGANISM: Dianthus sp. <220>FEATURE: <223> OTHER INFORMATION: DHS <400> SEQUENCE: 33 Arg Ser Ile LysCys Leu Ala Pro Thr Phe Lys Gly Asp Phe Ala Leu 1 5 10 15 Pro Gly AlaGln Leu Arg Ser Lys Gly Leu Asn Arg Ile Gly Asn Leu 20 25 30 Leu Val ProAsn Asp Asn Tyr Cys Lys Phe Glu Asp Trp Ile Ile Pro 35 40 45 Ile Leu AspLys Met Leu Glu Glu Gln Ile Ser Glu Lys Ile Leu Trp 50 55 60 Thr Pro SerLys Leu Ile Gly Arg Leu Gly Arg Glu Ile Asn Asp Glu 65 70 75 80 Ser SerTyr Leu Tyr Trp Ala Phe Lys Asn Asn Ile Pro Val Phe Cys 85 90 95 Pro GlyLeu Thr Asp Gly Ser Leu Gly Asp Met Leu Tyr Phe His Ser 100 105 110 PheArg Asn Pro Gly Leu Ile Ile Asp Val Val Gln Asp Ile Arg Ala 115 120 125Val Asn Gly Glu Ala Val His Ala Ala Pro Arg Lys Thr Gly Met Ile 130 135140 Ile Leu Gly Gly Gly Leu Pro Lys His His Ile Cys Asn Ala Asn Met 145150 155 160 Met Arg Asn Gly Ala Asp Tyr Ala Val Phe Ile Asn Thr 165 170<210> SEQ ID NO 34 <211> LENGTH: 37 <212> TYPE: PRT <213> ORGANISM:Arabidopsis sp. <220> FEATURE: <223> OTHER INFORMATION: DHS <400>SEQUENCE: 34 Ala Arg Pro Asp Glu Ala Val Ser Trp Gly Lys Ile Arg Gly SerAla 1 5 10 15 Lys Thr Val Lys Val Cys Phe Leu Ile Ser Ser His Pro AsnLeu Tyr 20 25 30 Leu Thr Gln Trp Phe 35 <210> SEQ ID NO 35 <211> LENGTH:52 <212> TYPE: PRT <213> ORGANISM: Lycopersicon sp. <220> FEATURE: <223>OTHER INFORMATION: DHS <400> SEQUENCE: 35 Gly Ala Arg Pro Asp Glu AlaVal Ser Trp Gly Lys Ile Arg Gly Gly 1 5 10 15 Ala Lys Thr Val Lys ValHis Cys Asp Ala Thr Ile Ala Phe Pro Ile 20 25 30 Leu Val Ala Glu Thr PheAla Ala Lys Ser Lys Glu Phe Ser Gln Ile 35 40 45 Arg Cys Gln Val 50

What is claimed is:
 1. An isolated DNA molecule encodingsenescence-induced deoxyhypusine synthase, wherein the DNA moleculehybridizes under low stringency conditions with SEQ ID NO:1 and/or SEQID NO:9, or a functional derivative of the isolated DNA molecule whichhybridizes with SEQ ID NO:1 and/or SEQ ID NO: 9 with the proviso thatthe DNA molecule does not have the sequence of SEQ ID NO:5.
 2. Theisolated DNA molecule of claim 1 wherein the DNA molecule has thenucleotide sequence of SEQ ID NO:1 or SEQ ID NO:9.
 3. An isolatedsenescence-induced deoxyhypusine synthase encoded by a nucleotidesequence which hybridizes under low stringency conditions with SEQ IDNO:1 and/or SEQ ID NO:9, or a functional derivative of thesenescence-induced deoxyhypusine synthase.
 4. The senescence-induceddeoxyhypusine synthase of claim 3 wherein the deoxyhypusine synthase hasthe amino acid sequence SEQ ID NO:2 or SEQ ID NO:10.
 5. An isolated DNAmolecule encoding senescence-induced elF-5A, wherein the DNA moleculehybridizes under low stringency conditions with SEQ ID NO:11, SEQ IDNO:13 and/or SEQ ID NO:15, or a functional derivative of the isolatedDNA molecule which hybridizes with SEQ ID NO:11, SEQ ID NO:13 and/or SEQID NO:15.
 6. The isolated DNA molecule of claim 5 wherein the DNAmolecule has the nucleotide sequence of SEQ ID NO:11, SEQ ID NO:13 orSEQ ID NO:15.
 7. A vector for transformation of plant cells comprising(a) antisense nucleotide sequences substantially complementary to (1) acorresponding portion of one strand of a DNA molecule encodingsenescence-induced deoxyhypusine synthase wherein the DNA moleculeencoding senescence-induced deoxyhypusine synthase hybridizes under lowstringency conditions with SEQ ID NO:1, SEQ ID NO:5 and/or SEQ ID NO:9or (2) a corresponding portion of an RNA sequence encoded by the DNAmolecule encoding senescence-induced deoxyhypusine synthase; and (b)regulatory sequences operatively linked to the antisense nucleotidesequences such that the antisense nucleotide sequences are expressed ina plant cell into which it is transformed.
 8. The vector according toclaim 7 wherein the regulatory sequences comprise a promoter and atranscription termination region.
 9. The vector according to claim 7wherein the regulatory sequences comprise a constitutive promoter. 10.The vector according to claim 7 wherein the regulatory sequencescomprise a plant tissue-specific promoter.
 11. The vector according toclaim 7 wherein the regulatory sequences comprise a senescence-inducedplant promoter.
 12. The vector according to claim 7 wherein theregulatory sequences comprise a viral promoter.
 13. The vector accordingto claim 12 wherein the regulatory sequences further comprise aconstitutive promoter.
 14. The vector according to claim 7 furthercomprising (a) antisense nucleotide sequences substantiallycomplementary to (1) a corresponding portion of one strand of a DNAmolecule encoding senescence-induced elF-5A wherein the DNA moleculeencoding senescence-induced elF-5A hybridizes under low stringencyconditions with SEQ ID NO:11, SEQ ID NO:13 and/or SEQ ID NO:15 or (2) acorresponding portion of an RNA sequence encoded by the DNA moleculeencoding senescence-induced elF-5A; and (b) regulatory sequencesoperatively linked to the antisense nucleotide sequences such that theantisense nucleotide sequences are expressed in a plant cell into whichit is transformed.
 15. A vector for transformation of plant cellscomprising (a) antisense nucleotide sequences substantiallycomplementary to (1) a corresponding portion of one strand of a DNAmolecule encoding senescence-induced elF-5A wherein the DNA moleculeencoding senescence-induced elF-5A hybridizes under low stringencyconditions with SEQ ID NO:11, SEQ ID NO:13 and/or SEQ ID NO:15 or (2) acorresponding portion of an RNA sequence encoded by the DNA moleculeencoding senescence-induced elF-5A; and (b) regulatory sequencesoperatively linked to the nucleotide sequences such that the antisensenucleotide sequences are expressed in a plant cell into which it istransformed.
 16. An antisense oligonucleotide or polynucleotide encodingan RNA molecule which is substantially complementary to (i) acorresponding portion of an RNA transcript of a plant senescence-induceddeoxyhypusine synthase gene, wherein said plant gene hybridizes underlow stringency conditions with SEQ ID NO:1, SEQ ID NO:5 and/or SEQ IDNO:9 or (ii) a corresponding portion of an RNA transcript of a plantsenescence-induced elF-5A gene, wherein said plant gene hybridizes underlow stringency conditions with SEQ ID NO:11, SEQ ID NO:13 and/or SEQ IDNO:15.
 17. The antisense oligonucleotide or polynucleotide according toclaim 16 wherein the oligonucleotide or polynucleotide comprises aboutsix to about 100 nucleotides.
 18. The antisense oligonucleotide orpolynucleotide according to claim 16 wherein the antisenseoligonucleotide or polynucleotide is substantially complementary to acorresponding portion of the 5′-non-coding region of the RNA transcript.19. The antisense oligonucleotide or polynucleotide according to claim16 wherein the antisense oligonucleotide or polynucleotide issubstantially complementary to a corresponding portion of the 3′-end ofthe RNA transcript.
 20. The antisense oligonucleotide or polynucleotideaccording to claim 16 wherein the antisense oligonucleotide orpolynucleotide is substantially complementary to the 3′-end ofArabidopsis senescence-induced DHS gene.
 21. The antisenseoligonucleotide or polynucleotide according to claim 19 wherein theantisense oligonucleotide or polynucleotide is substantiallycomplementary to SEQ ID NO:23.
 22. The antisense oligonucleotide orpolynucleotide according to claim 19 wherein the antisenseoligonucleotide or polynucleotide is substantially complementary to SEQID NO:30.
 23. A vector comprising a DNA molecule encoding (a)senescence-induced deoxyhypusine synthase, wherein the DNA moleculehybridizes under low stringency conditions with SEQ ID NO:1, SEQ IDNO:5, and/or SEQ ID NO:9; and (b) regulatory sequences operativelylinked to the DNA molecule such that the deoxyhypusine synthase isexpressed in a plant cell into which it is transformed.
 24. A vectorcomprising a DNA molecule encoding (a) senescence-induced elF-5A,wherein the DNA molecule hybridizes under low stringency conditions withSEQ ID NO:11, SEQ ID NO:13, and/or SEQ ID NO:15; and (b) regulatorysequences operatively linked to the DNA molecule such that elF-5A isexpressed in a plant cell into which it is transformed.
 25. A vectorcomprising a DNA molecule encoding (a) senescence-induced deoxyhypusinesynthase, wherein the DNA molecule hybridizes under low stringencyconditions with SEQ ID NO:1, SEQ ID NO:5, and/or SEQ ID NO:9; (b)senescence-induced elF-5A, wherein the DNA molecule hybridizes under lowstringency conditions with SEQ ID NO:11, SEQ ID NO:13, and/or SEQ IDNO:15; and (b) regulatory sequences operatively linked to the DNAmolecule such that the senescence-induced deoxhypusine synthase and theelF-5A are expressed in a plant cell into which it is transformed.
 26. Abacterial cell transformed with the vector according to any one ofclaims 7, 14 or
 15. 27. A plant cell transformed with the vectoraccording to any one of claims 7, 14 or
 15. 28. A plant and progenythereof, wherein the plant is generated from a plant cell transformedwith the vector according to any one of claims 7, 14 or
 15. 29. A plantpart derived from a plant or progeny according to claim
 26. 30. A methodfor inhibiting the expression of endogenous senescence-induceddeoxyhypusine synthase, elF-5A or both in a plant, said methodcomprising (1) integrating into the genome of the plant a vectorcomprising (A) antisense nucleotide sequences substantiallycomplementary to (i) a corresponding portion of one strand of a DNAmolecule encoding the endogenous senescence-induced deoxyhypusinesynthase, wherein the DNA molecule encoding the endogenoussenescence-induced deoxyhypusine synthase hybridizes with SEQ ID NO:1,SEQ ID NO:5, and/or SEQ ID NO:9 or (ii) a corresponding portion of anRNA sequence encoded by the endogenous senescence-induced deoxyhypusinesynthase gene, (iii) a corresponding portion of one strand of a DNAmolecule encoding the endogenous senescence-induced elF-5A, wherein theDNA molecule encoding the endogenous senescence-induced deoxyhypusinesynthase hybridizes with SEQ ID NO:11, SEQ ID NO:13, and/or SEQ IDNO:15, (iv) a corresponding portion of an RNA sequence encoded by theendogenous senescence-induced elF-5A, or (v) a combination of (I) or(ii) and (iii) or (iv); and (B) regulatory sequences operatively linkedto the antisense nucleotide sequences such that the antisense nucleotidesequences are expressed; and (2) growing said plant, whereby saidantisense nucleotide sequences are transcribed and bind to said RNAsequence, whereby expression of the senescence-induced deoxyhypusinesynthase gene, senescence-induced elF-5A gene or both is inhibited. 31.The method according to claim 30 wherein the portion of the DNA or theportion of the RNA to which the antisense nucleotide sequence issubstantially complementary comprises 5′-non-coding or 3′-coding and/ornon-coding sequences.
 32. The method according to claim 30 wherein theantisense nucleotide sequence is substantially complementary to SEQ IDNO:23
 33. The method according to claim 30 wherein the antisensenucleotide sequence is substantially complementary to SEQ ID NO:30. 34.The method according to claim 30 wherein said inhibition results inaltered senescence of the plant.
 35. The method according to claim 30wherein said inhibition results in increased resistance of said plant toenvironmental stress-induced and/or pathogen-induced senescence.
 36. Themethod according to claim 30 wherein said inhibition results inincreased biomass of said plant.
 37. The method according to claim 30wherein said inhibition results in delayed fruit softening and spoilagein said plant.
 38. The method according to claim 30 wherein saidinhibition results in increased seed yield from said plant.
 39. Themethod according to claim 30 wherein the regulatory sequences comprise aconstitutive promoter active in the plant.
 40. The method according toclaim 30 wherein the regulatory sequences comprise a tissue specificpromoter active in the plant.
 41. The method according to claim 30wherein the regulatory sequences comprise a senescence-induced promoteractive in the plant.
 42. The method according to claim 30 wherein saidplant is selected from the group consisting of fruit bearing plants,flowering plants, vegetables, agronomic crop plants and forest species.43. The method according to claim 30 wherein the plant is a tomato. 44.The method according to claim 30 wherein the plant is a flowering plant.45. A method for inhibiting the expression of an endogenoussenescence-induced deoxyhypusine synthase gene in a plant cell, saidmethod comprising (1) integrating into the genome of at least one cellof the plant a vector comprising (A) an isolated DNA molecule encodingexogenous senescence-induced deoxyhypusine synthase, wherein the DNAmolecule hybridizes under low stringency conditions with SEQ ID NO:1,SEQ ID NO:5, and/or SEQ ID NO:9 or a functional derivative of theisolated DNA molecule which hybridizes with SEQ ID:1, SEQ ID NO:5,and/or SEQ ID NO:9; and (B) regulatory sequences operatively linked tothe DNA molecule such that the exogenous senescence-induceddeoxyhypusine synthase encoded thereby is expressed; and (2) growingsaid plant, whereby said DNA molecule is over-expressed and theendogenous senescence-induced deoxyhypusine synthase gene is inhibitedby exogenous senescence-induced deoxyhypusine synthase.
 46. The methodaccording to claim 45 wherein the regulatory sequences comprise aconstitutive promoter.
 47. A method of altering age-related senescenceand/or environmental stress-related senescence in a plant, said methodcomprising (1) integrating into the genome of the plant a vectorcomprising (A) antisense nucleotide sequences substantiallycomplementary to (I) a corresponding portion of one strand of a DNAmolecule encoding the endogenous senescence-induced deoxyhypusinesynthase, wherein the DNA molecule encoding the endogenoussenescence-induced deoxyhypusine synthase hybridizes with SEQ ID NO:1,SEQ ID NO: 5 and/or SEQ ID NO:9 or (ii) at least a portion of an RNAsequence encoded by the endogenous senescence-induced deoxyhypusinesynthase gene, (iii) a corresponding portion of one strand of a DNAmolecule encoding the endogenous senescence-induced elF-5A gene, whereinthe DNA molecule encoding the endogenous senescence-induced elF-5Ahybridizes with SEQ ID NO:11, SEQ ID NO:13 and/or SEQ ID NO:15, (iv) acorresponding portion of an RNA sequence encoded by the endogenoussenescence-induced elF-5A gene, or (v) a combination of (I) or (ii) and(iii) or (iv); (B) regulatory sequences operatively linked to theantisense nucleotide sequences such that the antisense nucleotidesequences are expressed; and (2) growing said plant, whereby saidantisense nucleotide sequences are transcribed and bind to said RNAsequence, whereby expression of said senescence-induced deoxyhypusinesynthase gene, senescence-induced elF-5A gene or both is inhibited. 48.A transgenic plant cell comprising a vector according to any one ofclaims 7, 14, 15 or a combination of said vectors.
 49. A transgenicplant cell comprising a vector according to any one of claims 23, 24, 25or a combination of said vectors.
 50. A plasmid comprising a replicationsystem functional in a prokaryotic host and an antisense oligonucleotideor polynucleotide according to claim
 16. 51. A plasmid comprising areplication system functional in Agrobacterium and an antisenseoligonucleotide or polynucleotide according to claim
 16. 52. A plant andprogeny thereof, wherein said plant is derived from a cell havinginhibited or reduced expression of senescence-induced deoxyhypusinesynthase, senescence-induced elF-5A or both, said cell comprising avector according to any one of claims 7, 14 or
 15. 53. A plant andprogeny thereof, wherein said plant is derived from a cell havinginhibited or reduced expression of senescence-induced deoxyhypusinesynthase, senescence-induced elF-5A, or both, wherein said cell isproduced by (1) integrating into the genome of the cell a vectorcomprising (A) antisense nucleotide sequences substantiallycomplementary to (i) a corresponding portion of one strand of a DNAmolecule encoding the endogenous senescence-induced deoxyhypusinesynthase, wherein the DNA molecule encoding the endogenoussenescence-induced deoxyhypusine synthase hybridizes with SEQ ID NO:1,SEQ ID NO: 5, and/or SEQ ID NO:9 or (ii) a corresponding portion of anRNA sequence encoded by the endogenous senescence-induced deoxyhypusinesynthase gene, (iii) a corresponding portion of one strand of a DNAmolecule encoding the endogenous senescence-induced elF-5A gene, whereinthe DNA molecule encoding the endogenous senescence-induced elF-5Ahybridizes with SEQ ID NO:11, SEQ ID NO:13 and/or SEQ ID NO:15, (iv) acorresponding portion of an RNA sequence encoded by the endogenoussenescence-induced elF-5A gene, or (v) a combination of (I) or (ii) and(iii) or (iv); and (B) regulatory sequences operatively linked to theantisense nucleotide sequences such that the antisense nucleotides areexpressed; and (2) growing said cell, whereby said antisense nucleotidesequences are transcribed and bind to said RNA sequence, wherebyexpression of said senescence-induced deoxyhypusine synthase gene,senescence-induced elF-5A gene or both is inhibited.
 54. The plant andprogeny according to claim 53 wherein the plant is a tomato.
 55. Theplant and progeny according to claim 54 wherein the plant is a floweringplant.
 56. A method of inhibiting seed aging, said method comprising (1)integrating into the genome of a plant a vector comprising (A) antisensenucleotide sequences substantially complementary to (i) a correspondingportion of one strand of a DNA molecule encoding an endogenousaging-induced deoxyhypusine synthase, wherein DNA encoding saidendogenous aging-induced deoxyhypusine synthase hybridizes with SEQ IDNO:1, SEQ ID NO:5 and/or SEQ ID NO:9 or (ii) a corresponding portion ofan RNA sequence transcribed from a DNA molecule encoding an endogenoussenescence-induced deoxyhypusine synthase gene; and (B) regulatorysequences operatively linked to the antisense nucleotide sequences; and(2) growing said plant, whereby said antisense nucleotide sequences aretranscribed and bind to said RNA sequence and expression of saidaging-induced deoxyhypusine synthase gene is inhibited.
 57. The methodaccording to claim 56 further comprising integrating into the genome ofa plant a vector comprising (A) antisense nucleotide sequencessubstantially complementary to (i) a corresponding portion of one strandof a DNA molecule encoding an endogenous aging-induced elF-5A gene,wherein DNA encoding said endogenous aging-induced elF-5A hybridizeswith SEQ ID NO:11, SEQ ID NO:13 and/or SEQ ID NO:15 or (ii) acorresponding portion of an RNA sequence transcribed from a DNA moleculeencoding an endogenous senescence-induced elF-5A gene; and (B)regulatory sequences operatively linked to the antisense nucleotidesequences.
 58. A method of inhibiting seed aging, said method comprising(1) integrating into the genome of a plant a vector comprising (A)antisense nucleotide sequences substantially complementary to (i) acorresponding portion of one strand of a DNA molecule encoding anendogenous aging-induced deoxyhypusine synthase, wherein DNA encodingsaid endogenous aging-induced deoxyhypusine synthase hybridizes with SEQID NO:1, SEQ ID NO:5 and/or SEQ ID NO:9 or (ii) a corresponding portionof a substantially complementary RNA sequence transcribed from a DNAmolecule encoding an endogenous senescence-induced deoxyhypusinesynthase gene, (iii) a corresponding portion of one strand of a DNAmolecule encoding an endogenous aging-induced elF-5A gene, wherein DNAencoding said endogenous aging-induced elF-5A hybridizes with SEQ IDNO:11, SEQ ID NO:13 and/or SEQ ID NO:15, (iv) a corresponding portion ofa substantially complementary RNA sequence transcribed from a DNAmolecule encoding an endogenous senescence-induced elF-5A gene; or (v) acombination of (i) or (ii) and (iii) or (iv); and (B) regulatorysequences operatively linked to the antisense nucleotide sequences; and(2) growing said plant, whereby said antisense nucleotide sequences aretranscribed and bind to said substantially complementary RNA sequenceand expression of said aging-induced deoxyhypusine synthase gene, elF-5Agene or both is inhibited.
 59. A method of increasing seed yield from aplant, said method comprising (1) integrating into the genome of theplant a vector comprising (A) antisense nucleotide sequencessubstantially complementary to (i) a corresponding portion of one strandof a DNA molecule encoding an endogenous senescence-induceddeoxyhypusine synthase, wherein DNA encoding said endogenoussenescing-induced deoxyhypusine synthase hybridizes with SEQ ID NO:1,SEQ ID NO:5 and/or SEQ ID NO:9 or (ii) a corresponding portion of an RNAsequence transcribed from a DNA molecule encoding an endogenoussenescence-induced deoxyhypusine synthase gene; and (B) regulatorysequences operatively linked to the antisense nucleotide sequences; and(2) growing said plant, whereby said antisense nucleotide sequences aretranscribed and bind to said RNA sequence and expression of saiddeoxyhypusine synthase gene is inhibited.
 60. The method according toclaim 59 further comprising integrating into the genome of a plant avector comprising (A) antisense nucleotide sequences substantiallycomplementary to (i) a corresponding portion of one strand of a DNAmolecule encoding an endogenous aging-induced elF-5A gene, wherein DNAencoding said endogenous aging-induced elF-5A hybridizes with SEQ IDNO:11, SEQ ID NO:13 and/or SEQ ID NO:15 or (ii) a corresponding portionof an RNA sequence transcribed from a DNA molecule encoding anendogenous senescence-induced elF-5A gene; and (B) regulatory sequencesoperatively linked to the antisense nucleotide sequences.